Artificial cell death polypeptide for chimeric antigen receptor and uses thereof

ABSTRACT

Provided are polynucleotides encoding inactivated cell surface receptors. Also provided are genetically engineered induced pluripotent stem cells (iPSCs) and derivative cells thereof expressing a chimeric antigen receptor (CAR) and methods of using the same. Also provided are compositions, polypeptides, vectors, and methods of manufacturing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 63/120,948 filed Dec. 3, 2020, the disclosure of whichis incorporated by reference herein in its entirety.

TECHNICAL FIELD

This application provides genetically engineered induced pluripotentstem cells (iPSCs) and derivative cells thereof. Also provided are usesof the iPSCs or derivative cells thereof to express a chimeric antigenreceptor for allogenic cell therapy. Also provided are related vectors,polynucleotides, and pharmaceutical compositions.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “066461-3US2_Sequence Listing” and a creation date of Nov. 1,2021, and having a size of 113 kb. The sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND

Chimeric antigen receptors (CARs) significantly enhance anti-tumoractivity of immune effector cells. CARs are engineered receptorstypically comprising an extracellular targeting domain that is linked toa linker peptide, a transmembrane (TM) domain, and one or moreintracellular signaling domains. Traditionally, the extracellular domainconsists of an antigen binding fragment of an antibody (such as a singlechain Fv, scFv) that is specific for a given tumor-associated antigen(TAA) or cell surface target. The extracellular domain confers the tumorspecificity of the CAR, while the intracellular signaling domainactivates the T cell that has been genetically engineered to express theCAR upon TAA/target engagement. The engineered immune effector cells arere-infused into cancer patients, where they specifically engage and killcells expressing the TAA target of the CAR (Maus et al., Blood. 2014Apr. 24; 123(17):2625-35; Curran and Brentjens, J Clin Oncol. 2015 May20; 33(15):1703-6).

Autologous, patient-specific CAR-T therapy has emerged as a powerful andpotentially curative therapy for cancer, especially for CD19-positivehematological malignancies. However, the autologous T cells must begenerated on a custom-made basis, which remains a significant limitingfactor for large-scale clinical application due to the production costsand the risk of production failure. The development of CAR-T technologyand its wider application is also limited due to a number of other keyshortcomings, including, e.g., a) an inefficient anti-tumor response insolid tumors, b) limited penetration and susceptibility of adoptivelytransferred CAR T cells to an immunosuppressive tumor microenvironment(TME), c) poor persistence of CAR-T cells in vivo, d) serious adverseevents in the patients including cytokine release syndrome (CRS) andgraft-versus-host disease (GVHD) mediated by the CAR-T, and e) the timerequired for manufacturing.

Therefore, there is an unmet need for therapeutically sufficient andfunctional antigen-specific immune cells for effective use inimmunotherapy.

BRIEF SUMMARY

In one general aspect, provided is a polynucleotide encoding anartificial cell death polypeptide. In certain embodiments, thepolynucleotide encodes an inactivated cell surface receptor thatcomprises a monoclonal antibody-specific epitope and an interleukin 15(IL-15), wherein the inactivated cell surface receptor and the IL-15 areoperably linked by an autoprotease peptide.

In certain embodiments, the inactivated cell surface receptor isselected from the group of monoclonal antibody specific epitopesselected from epitopes specifically recognized by ibritumomab, tiuxetan,muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin,cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumabpegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab,omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab,trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab,golimumab, ipilimumab, ofatumumab, panitumumab, and ustekinumab.

In certain embodiments, the inactivated cell surface receptor is atruncated epithelial growth factor receptor (tEGFR) variant.

In certain embodiments, the autoprotease peptide comprises or is aporcine tesehovirus-1 2A (P2A) peptide.

In certain embodiments, the tEGFR variant consists of an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to SEQ ID NO: 71, preferably SEQ ID NO:71.

In certain embodiments, the IL-15 comprises an amino acid sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO: 72, preferably SEQ ID NO: 72.

In certain embodiments, the autoprotease peptide comprises an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to SEQ ID NO: 73, preferably SEQ ID NO:73.

In certain embodiments, the polynucleotide consists of operably linkedpolynucleotides encoding a truncated epithelial growth factor receptor(tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, anautoprotease peptide having the amino acid sequence of SEQ ID NO: 73,and an interleukin 15 (IL-15) having the amino acid sequence of SEQ IDNO: 72.

Also provided is a polynucleotide encoding an inactivated cell surfacereceptor that comprises an epitope specifically recognized by anantibody selected from the group consisting of cetuximab, matuzumab,necitumumab, panitumumab, polatuzumab vedotin, rituximab andtrastuzumab, and an IL-15, wherein the epitope and the cytokine areoperably linked by a P2A sequence.

In certain embodiments, the inactivated cell surface receptor comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:74, 79, 81, and 83.

Also provided is a protein encoded by a polynucleotide of theapplication.

Also provided is an induced pluripotent stem cell (iPSC) or a derivativecell thereof comprising a polynucleotide of the application

Also provided is a vector comprising a polynucleotide of theapplication.

In certain embodiments, the vector further comprises:

(i) a promoter;

(ii) a terminator and/or a polyadenylation signal sequence;

(iii) a left homology sequence; and

(iv) a right homology sequence.

In certain embodiments, the left homology sequence comprises apolynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to the polynucleotidesequence of SEQ ID NO: 84.

In certain embodiments, the right homology sequence comprises apolynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to the polynucleotidesequence of SEQ ID NO: 85.

In certain embodiments, the vector comprises a polynucleotide sequencehaving at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% sequence identity to SEQ ID NO: 86.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofpreferred embodiments of the present application, will be betterunderstood when read in conjunction with the appended drawings. Itshould be understood, however, that the application is not limited tothe precise embodiments shown in the drawings.

FIGS. 1A-1C show schematics of vectors (plasmids) according toembodiments of the application. FIG. 1A shows CIITA targeting transgeneplasmid with a CMV early enhancer/chicken β actin (CAG) promoter, SV40terminator/polyadenylation signal, and tEGFR-IL15 coding sequence. FIG.1B shows AAVS1 targeting transgene plasmid with a CAG promoter, SV40terminator/polyadenylation, and anti-CD19 scFv chimeric antigen receptor(CAR) coding sequence. FIG. 1C shows B2M targeting transgene plasmidwith a CAG promoter, SV40 terminator/polyadenylation, andPeptide-B2M-HLA-E coding sequence.

FIG. 2 shows a graph demonstrating CAR-iNK cell-mediated target cellcytotoxicity over time in Reh cells and CD19 knockout (CD19KO) Rehcells.

FIGS. 3A-C show functionality of iNK cell expressing CAR-IL15 comparedto iNK cells expressing CAR alone. FIG. 3A shows a graph demonstratingIL-15 concentration (pg/ml/1e6 cells/24 hours) released from CAR iNKcells and CAR/IL15 iNK cells. FIG. 3B shows graphs demonstratingpercentage iNK cells after 20 days in the blood and lungs of miceinjected with CAR iNK cells or CAR-IL15 iNK cells. FIG. 3C shows graphsdemonstrating percentage iNK cells in the lungs of mice injected withCAR iNK cells or CAR-IL15 iNK cells and with and without recombinantIL-15.

FIGS. 4A-C show the proliferation and serial killing of CAG-CAR-IL-15iNK cells. FIG. 4A shows a graph demonstrating the serial killing ofCD19+ Reh cells by CAG-CAR/IL15-iNK cells over time. FIG. 4B shows agraph demonstrating the increased proliferation of CAG-CAR/IL-15 iNKcells compared to CAG-CAR iNK cells. FIG. 4C shows a graph demonstratingthe increased target serial killing of CD19+ Raji cells over time byCAG-CAR/IL-15 iNK cells compared to CAG-CAR iNK cells.

FIGS. 5A-5B show cytotoxicity of CAG-CAR-IL15 expressing iNK cells withand without human recombinant IL12. FIG. 5A shows a graph demonstratingRaji cell death overtime when cultured with CAG-CAR-IL15 iNK cells withand without IL12. FIG. 5B shows a graph demonstrating tumor growthmeasured as mean whole body luminescent average radiance of mice infusedwith IL12-primed and unprimed CAG-CAR-IL15 iNK cells.

FIGS. 6A-6B show Cetuximab-induced cell elimination in CAG-CARexpressing iNK cells and CAG-CAR-IL15-tEGFR expressing iNK cells. FIG.6A shows a graph demonstrating percentage Annexin-V staining in CAG-CARexpressing cells. FIG. 6B shows a graph demonstrating percentageAnnexin-V staining in CAG-CAR-IL15-tEGFR expressing cells.

FIG. 7A shows an Incucyte based assay measuring the loss of Nuclight RedK562 cells over time with an effector to target ratio of 20:1.Normalized target cell count as a percentage of target cell only countfor four iNK1248-iPSC611 and the average of 3 PB-NKs. Each data point isthe average of 3 replicates and error bars represent standard error ofthe mean.

FIG. 7B shows an Incucyte based assay measuring the loss of Nuclight RedK562 cells over time with an effector to target ratio of 10:1.Normalized target cell count as a percentage of target cell only countfor four iNK1248-iPSC611 and the average of 3 PB-NKs. Each data point isthe average of 3 replicates and error bars represent standard error ofthe mean.

FIG. 7C shows an Incucyte based assay measuring the loss of Nuclight RedK562 cells over time with an effector to target ratio of 1:1. Normalizedtarget cell count as a percentage of target cell only count for fouriNK1248-iPSC611 and the average of 3 PB-NKs. Each data point is theaverage of 3 replicates and error bars represent standard error of themean.

FIG. 8 shows a flow-based NK purity check of PB-NKs isolated from threePBMC donors and iNK1248-iPSC611.

FIG. 9A shows an Incucyte based assay measuring the loss of Nuclight Redtarget cells over time with an effector to target ratio of 10:1.Normalized target cell count in Reh and Reh-CD19KO co-cultured withiNK1248-iPSC611 at an effector to target ratio of 10:1 as a percentageof target cell only counts. Each data point is the average of 3replicates and error bars represent standard error of the mean.

FIG. 9B shows an Incucyte based assay measuring the loss of Nuclight Redtarget cells over time with an effector to target ratio of 5:1.Normalized target cell count in Reh and Reh-CD19KO co-cultured withiNK1248-iPSC611 at an effector to target ratio of 5:1 as a percentage oftarget cell only counts. Each data point is the average of 3 replicatesand error bars represent standard error of the mean.

FIG. 9C shows an Incucyte based assay measuring the loss of Nuclight Redtarget cells over time with an effector to target ratio of 1:1.Normalized target cell count in Reh and Reh-CD19KO co-cultured withiNK1248-iPSC611 at an effector to target ratio of 1:1 as a percentage oftarget cell only counts. Each data point is the average of 3 replicatesand error bars represent standard error of the mean.

FIG. 9D shows an Incucyte based assay measuring the loss of Nuclight Redtarget cells over time with an effector to target ratio of 1:5.Normalized target cell count in Reh and Reh-CD19KO co-cultured withiNK1248-iPSC611 at an effector to target ratio of 1:5 as a percentage oftarget cell only counts. Each data point is the average of 3 replicatesand error bars represent standard error of the mean.

FIG. 10A shows an Incucyte based assay measuring the loss of NuclightRed target cells over time with an effector to target ratio of 10:1.Normalized target cell count in NALM6 and NALM6-CD19K0 co-cultured withiNK1248-iPSC611 at an effector to target ratio of 10:1 as a percentageof target cell only counts. Each data point is the average of 3replicates and error bars represent standard error of the mean.

FIG. 10B shows an Incucyte based assay measuring the loss of NuclightRed target cells over time with an effector to target ratio of 5:1.Normalized target cell count in NALM6 and NALM6-CD19KO co-cultured withiNK1248-iPSC611 at an effector to target ratio of 5:1 as a percentage oftarget cell only counts. Each data point is the average of 3 replicatesand error bars represent standard error of the mean.

FIG. 10C shows an Incucyte based assay measuring the loss of NuclightRed target cells over time with an effector to target ratio of 1:1.Normalized target cell count in NALM6 and NALM6-CD19KO co-cultured withiNK1248-iPSC611 at an effector to target ratio of 1:1 as a percentage oftarget cell only counts. Each data point is the average of 3 replicatesand error bars represent standard error of the mean.

FIG. 10D shows an Incucyte based assay measuring the loss of NuclightRed target cells over time with an effector to target ratio of 1:5.Normalized target cell count in NALM6 and NALM6-CD19KO co-cultured withiNK1248-iPSC611 at an effector to target ratio of 1:5 as a percentage oftarget cell only counts. Each data point is the average of 3 replicatesand error bars represent standard error of the mean.

FIG. 11 shows cumulative fold expansion of iNK1248-iPSC611 and WTiNK1487-iPSC005 over a 21-day persistence assay without exogenous IL2support. Cells were cultured in basal NKCM for 14 days at 37° C. with 5%CO2. Every 3-4 days, all conditions were harvested, counted on theViCell Blu, resuspended at 0.5e6/mL in appropriate media and thenreplated. After 21 days, cumulative fold change was calculated.

FIG. 12A shows cumulative fold expansion of iNK1248-iPSC611 and WTiNK1487-iPSC005 over a 21-day persistence assay. Cells were cultured inNKCM containing one of six IL2 concentrations: 10 nM for 21 days at 37°C. with 5% CO2. Every 3-4 days, all conditions were harvested, countedon the ViCell Blu, resuspended at 0.5e6/mL in appropriate media and thenreplated. After 21 days, cumulative fold change was calculated.

FIG. 12B shows cumulative fold expansion of iNK1248-iPSC611 and WTiNK1487-iPSC005 over a 21-day persistence assay. Cells were cultured inNKCM containing one of six IL2 concentrations: 3 nM for 21 days at 37°C. with 5% CO2. Every 3-4 days, all conditions were harvested, countedon the ViCell Blu, resuspended at 0.5e6/mL in appropriate media and thenreplated. After 21 days, cumulative fold change was calculated.

FIG. 12C shows cumulative fold expansion of iNK1248-iPSC611 and WTiNK1487-iPSC005 over a 21-day persistence assay. Cells were cultured inNKCM containing one of six IL2 concentrations: 1 nM for 21 days at 37°C. with 5% CO2. Every 3-4 days, all conditions were harvested, countedon the ViCell Blu, resuspended at 0.5e6/mL in appropriate media and thenreplated. After 21 days, cumulative fold change was calculated.

FIG. 12D shows cumulative fold expansion of iNK1248-iPSC611 and WTiNK1487-iPSC005 over a 21-day persistence assay. Cells were cultured inNKCM containing one of six IL2 concentrations: 0.3 nM for 21 days at 37°C. with 5% CO2. Every 3-4 days, all conditions were harvested, countedon the ViCell Blu, resuspended at 0.5e6/mL in appropriate media and thenreplated. After 21 days, cumulative fold change was calculated.

FIG. 12E shows cumulative fold expansion of iNK1248-iPSC611 and WTiNK1487-iPSC005 over a 21-day persistence assay. Cells were cultured inNKCM containing one of six IL2 concentrations: 0.1 nM for 21 days at 37°C. with 5% CO2. Every 3-4 days, all conditions were harvested, countedon the ViCell Blu, resuspended at 0.5e6/mL in appropriate media and thenreplated. After 21 days, cumulative fold change was calculated.

FIG. 12F shows cumulative fold expansion of iNK1248-iPSC611 and WTiNK1487-iPSC005 over a 21-day persistence assay. Cells were cultured inNKCM containing one of six IL2 concentrations: 0 nM for 21 days at 37°C. with 5% CO2. Every 3-4 days, all conditions were harvested, countedon the ViCell Blu, resuspended at 0.5e6/mL in appropriate media and thenreplated. After 21 days, cumulative fold change was calculated.

FIG. 13 shows a gating strategy for ADCC assays. Cells were gated onlymphocytes, followed by exclusion of doublets, followed by gating onCellTrace Violet (CTV)+ iNK, and finally on LIVE/DEAD™ Near-IR+ todetermine % of dead therapeutic iNK targets. FSC-A=forward scatter area,SSC-A=side scatter area, FSC-H=forward scatter height, CTV=CellTraceViolet, NIR=Near-IR.

FIG. 14 shows EGFR staining on therapeutic iNK cells. EGFR PE levels ontherapeutic iNK stained with EGFR (black histogram) compared withunstained therapeutic iNK (gray histogram) or an unedited WT iNK (dashedline).

FIG. 15 shows cetuximab-mediated ADCC of therapeutic iNK cells. Percentspecific cell lysis of therapeutic iNK cells mediated by Cetuximab(black triangles) compared with human IgG1 isotype control (opentriangles). IL-2 activated PBMC were co-cultured with therapeutic iNK ata 25:1 E:T ratio for 16 hours and percent specific cell death of iNKdetermined. Each data point is a mean of triplicate wells, errorbars±standard deviation.

FIG. 16 shows select sensitivity of WT iNK cells to anti-HLA-ABCAb-mediated complement cytotoxicity.

FIG. 17 shows a gating strategy for allo-evasion CTL cytotoxicity andactivation assays. Cells were gated on quantitation beads andlymphocytes. Within lymphocytes exclusion of doublets, followed bygating on LIVE/DEAD™ Near-IR negative, followed by CTV to identify iNKcells and TCRαβ to identify T cells. Within T cells, CD4-negative,CD8-positive cells, followed by CD25 to identify activated CD8+ T cells.Key assay parameters Quantitation beads, live iNK cells and activatedCD8+ T cells are indicated. FSC-A=forward scatter area, SSC-A=sidescatter area, FSC-H=forward scatter height, FSC-W=forward scatter width,L-D=LIVE/DEAD™ Near-IR, CTV=CellTrace Violet.

FIGS. 18A-B show CTL-mediated lysis of iNK cells. Assessment of specificiNK lysis by FACS. FIG. 18A shows gating on iNK and T cells. FIG. 18Bshows specific lysis of iNK cells co-cultured with CTL at 5:1 CTL:iNKratio. Each symbol represents one donor, open bar is the parentalwild-type iNK cells, and the shaded bar is the edited β2MKO iNK cells.

FIG. 19A shows activation of iNK-specific CTL in co-cultures. FIG. 19Ashows a histogram plot of CD25 expression of CD8+ T cells. Dashed lineindicates T cells cultured alone, the solid open histogram indicates Tcells co-cultured with parental wild-type iNK cells, and the shadedhistogram indicates T cells co-cultured with edited 132MKO iNK cells.

FIG. 19B show activation of iNK-specific CTL in co-cultures. FIG. 19Bshows frequencies of activated T cells in co-cultures with parental iNKcells (open bar), β2MKO iNK cells (shaded bar), or T cells alone with notargets (hatched bar). Each symbol represents one donor.

FIG. 20 shows a gating strategy for all-evasion cytotoxicity assays.Cells were gated on lymphocytes, followed by exclusion of doublets,followed by gating on CellTrace Violet (CTV)+ iNK, and finally onLIVE/DEAD™ Near-IR+ to determine % of dead iNK targets. FSC-A=forwardscatter area, SSC-A=side scatter area, FSC-H=forward scatter height,CTV=CellTrace Violet, NIR=Near-IR.

FIG. 21 shows HLA-E staining on therapeutic iNK cells. HLA-E=openhistogram, mouse IgG1 isotype control=gray filled histogram.

FIG. 22 shows NKG2A staining on PBMCs. PBMC samples were gated on viablelymphocytes (data not shown), followed by a gate on CD3-CD56+ cells (“NKcells”). Frequencies of NKG2A-expressing NK cells were then determinedbased on an FMO.

FIG. 23 shows cell death of therapeutic iNK cells (gray bars) relativeto WT (black bars) compared with iNK lacking β2M (white bars). Freshlythawed PBMC were co-cultured with therapeutic iNK at a 25:1 E:T ratio inthe presence of 10 ng/mL IL-15 for 72 hours and cell death of edited iNKrelative to WT determined. Each data point is a mean of triplicatewells.

FIG. 24 shows mean percent body weight change of untreated mice (•), ormice treated intravenously with iPSC611 at 10×10⁶ (▾) and 15×10⁶ (♦)(cryogenic) cells. Means are plotted where ≥50% of the treatment groupare present. Arrows represent dosing days.

FIG. 25 shows mean whole body average radiance of untreated mice (•),and mice treated intravenously weekly for three doses with iPSC611 at10×10⁶ (▾) and 15×10⁶ (♦) (cryogenic) cells. Groups are plotted untilDay 21, the last imaging timepoint where the untreated control groupremained and the timepoint at which % TGI was calculated. Arrowsrepresent dosing days.

FIG. 26 shows percent survival of NALM6-bearing mice treated withiPSC611. Mice were left untreated, or treated intravenously weekly forthree doses with iPSC611 at 10×10⁶ and 15×10⁶ cryogenic cells. Mice werehumanely euthanized when in moribund condition and exhibiting signs ofexcessive tumor burden, as a surrogate for survival.

FIG. 27 shows persistence of iPSC611 in lungs and blood of NALM6-bearingmice. Mice were left untreated, or received a single intravenous dose ofiPSC611 at 15×10⁶ cryogenic cells. One-week post-injection, lungs andblood were harvested for FACS analysis. Number of iNK per 100,000lymphocytes is plotted for individual mice (o), and average per grouprepresented by bars.

FIG. 28 shows mean percent body weight change of mice treatedintravenously with iPSC611 at 15×10⁶ cells receiving IP PBS (•), orcetuximab at 40 mg/kg (▪). FIG. 29 shows presence of iPSC611 in lungsand blood of NSG mice. Mice were left untreated (naïve), or received asingle intravenous dose of iPSC611 at 15×10⁶ cells on Day 1. On Days 2and 3, mice were treated IP with 20 mL/kg PBS (•), or 40 mg/kg cetuximab(▪). All mice received rhIL-2 on Days 1 and 3. On Day 5, lungs and bloodwere sampled and processed for FACS analysis and detection of iPSC611.There was a significant 96% reduction of iNK in lungs (p=0.0002) and 95%reduction of iNK in the blood (p=0.0321) of cetuximab-treated mice. Datais represented as the Number of iNK per 100,000 lymphocytes per mouse,with mean±SD plotted.

DETAILED DESCRIPTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this application pertains. Otherwise, certain termsused herein have the meanings as set forth in the specification.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise.

Unless otherwise stated, any numerical values, such as a concentrationor a concentration range described herein, are to be understood as beingmodified in all instances by the term “about.” Thus, a numerical valuetypically includes ±10% of the recited value. For example, aconcentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, aconcentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).As used herein, the use of a numerical range expressly includes allpossible subranges, all individual numerical values within that range,including integers within such ranges and fractions of the values unlessthe context clearly indicates otherwise.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the application described herein. Such equivalents areintended to be encompassed by the application.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers and are intended to be non-exclusive or open-ended.For example, a composition, a mixture, a process, a method, an article,or an apparatus that comprises a list of elements is not necessarilylimited to only those elements but can include other elements notexpressly listed or inherent to such composition, mixture, process,method, article, or apparatus. Further, unless expressly stated to thecontrary, “or” refers to an inclusive or and not to an exclusive or. Forexample, a condition A or B is satisfied by any one of the following: Ais true (or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

As used herein, the conjunctive term “and/or” between multiple recitedelements is understood as encompassing both individual and combinedoptions. For instance, where two elements are conjoined by “and/or,” afirst option refers to the applicability of the first element withoutthe second. A second option refers to the applicability of the secondelement without the first. A third option refers to the applicability ofthe first and second elements together. Any one of these options isunderstood to fall within the meaning, and therefore satisfy therequirement of the term “and/or” as used herein. Concurrentapplicability of more than one of the options is also understood to fallwithin the meaning, and therefore satisfy the requirement of the term“and/or.”

As used herein, the term “consists of,” or variations such as “consistof” or “consisting of,” as used throughout the specification and claims,indicate the inclusion of any recited integer or group of integers, butthat no additional integer or group of integers can be added to thespecified method, structure, or composition.

As used herein, the term “consists essentially of,” or variations suchas “consist essentially of” or “consisting essentially of,” as usedthroughout the specification and claims, indicate the inclusion of anyrecited integer or group of integers, and the optional inclusion of anyrecited integer or group of integers that do not materially change thebasic or novel properties of the specified method, structure orcomposition. See M.P.E.P. § 2111.03.

As used herein, “subject” means any animal, preferably a mammal, mostpreferably a human. The term “mammal” as used herein, encompasses anymammal. Examples of mammals include, but are not limited to, cows,horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs,monkeys, humans, etc., more preferably a human.

It should also be understood that the terms “about,” “approximately,”“generally,” “substantially,” and like terms, used herein when referringto a dimension or characteristic of a component of the preferredinvention, indicate that the described dimension/characteristic is not astrict boundary or parameter and does not exclude minor variationstherefrom that are functionally the same or similar, as would beunderstood by one having ordinary skill in the art. At a minimum, suchreferences that include a numerical parameter would include variationsthat, using mathematical and industrial principles accepted in the art(e.g., rounding, measurement or other systematic errors, manufacturingtolerances, etc.), would not vary the least significant digit.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences (e.g., CAR polypeptides andthe CAR polynucleotides that encode them), refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et at, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.

Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions.

As used herein, the term “isolated” means a biological component (suchas a nucleic acid, peptide, protein, or cell) has been substantiallyseparated, produced apart from, or purified away from other biologicalcomponents of the organism in which the component naturally occurs,i.e., other chromosomal and extrachromosomal DNA and RNA, proteins,cells, and tissues. Nucleic acids, peptides, proteins, and cells thathave been “isolated” thus include nucleic acids, peptides, proteins, andcells purified by standard purification methods and purification methodsdescribed herein. “Isolated” nucleic acids, peptides, proteins, andcells can be part of a composition and still be isolated if thecomposition is not part of the native environment of the nucleic acid,peptide, protein, or cell. The term also embraces nucleic acids,peptides and proteins prepared by recombinant expression in a host cellas well as chemically synthesized nucleic acids.

As used herein, the term “polynucleotide,” synonymously referred to as“nucleic acid molecule,” “nucleotides,” “nucleic acids,” or “polynucleicacids,” refers to any polyribonucleotide or polydeoxyribonucleotide,which can be unmodified RNA or DNA or modified RNA or DNA.“Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that can be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short nucleic acidchains, often referred to as oligonucleotides.

A “construct” refers to a macromolecule or complex of moleculescomprising a polynucleotide to be delivered to a host cell, either invitro or in vivo. A “vector,” as used herein refers to any nucleic acidconstruct capable of directing the delivery or transfer of a foreigngenetic material to target cells, where it can be replicated and/orexpressed. The term “vector” as used herein comprises the construct tobe delivered. A vector can be a linear or a circular molecule. A vectorcan be integrating or non-integrating. The major types of vectorsinclude, but are not limited to, plasmids, episomal vector, viralvectors, cosmids, and artificial chromosomes. Viral vectors include, butare not limited to, adenovirus vector, adeno-associated virus vector,retrovirus vector, lentivirus vector, Sendai virus vector, and the like.

By “integration” it is meant that one or more nucleotides of a constructis stably inserted into the cellular genome, i.e., covalently linked tothe nucleic acid sequence within the cell's chromosomal DNA. By“targeted integration” it is meant that the nucleotide(s) of a constructis inserted into the cell's chromosomal or mitochondrial DNA at apre-selected site or “integration site”. The term “integration” as usedherein further refers to a process involving insertion of one or moreexogenous sequences or nucleotides of the construct, with or withoutdeletion of an endogenous sequence or nucleotide at the integrationsite. In the case, where there is a deletion at the insertion site,“integration” can further comprise replacement of the endogenoussequence or a nucleotide that is deleted with the one or more insertednucleotides.

As used herein, the term “exogenous” is intended to mean that thereferenced molecule or the referenced activity is introduced into, ornon-native to, the host cell. The molecule can be introduced, forexample, by introduction of an encoding nucleic acid into the hostgenetic material such as by integration into a host chromosome or asnon-chromosomal genetic material such as a plasmid. Therefore, the termas it is used in reference to expression of an encoding nucleic acidrefers to introduction of the encoding nucleic acid in an expressibleform into the cell. The term “endogenous” refers to a referencedmolecule or activity that is present in the host cell in its nativeform. Similarly, the term when used in reference to expression of anencoding nucleic acid refers to expression of an encoding nucleic acidnatively contained within the cell and not exogenously introduced.

As used herein, a “gene of interest” or “a polynucleotide sequence ofinterest” is a DNA sequence that is transcribed into RNA and in someinstances translated into a polypeptide in vivo when placed under thecontrol of appropriate regulatory sequences. A gene or polynucleotide ofinterest can include, but is not limited to, prokaryotic sequences, cDNAfrom eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g.,mammalian) DNA, and synthetic DNA sequences. For example, a gene ofinterest may encode an miRNA, an shRNA, a native polypeptide (i.e. apolypeptide found in nature) or fragment thereof; a variant polypeptide(i.e. a mutant of the native polypeptide having less than 100% sequenceidentity with the native polypeptide) or fragment thereof; an engineeredpolypeptide or peptide fragment, a therapeutic peptide or polypeptide,an imaging marker, a selectable marker, and the like.

“Operably-linked” refers to the association of nucleic acid sequences ona single nucleic acid fragment so that the function of one is affectedby the other. For example, a promoter is operably-linked with a codingsequence or functional RNA when it is capable of affecting theexpression of that coding sequence or functional RNA (i.e., the codingsequence or functional RNA is under the transcriptional control of thepromoter). Coding sequences can be operably-linked to regulatorysequences in sense or antisense orientation.

The term “expression” as used herein, refers to the biosynthesis of agene product. The term encompasses the transcription of a gene into RNA.The term also encompasses translation of RNA into one or morepolypeptides, and further encompasses all naturally occurringpost-transcriptional and post-translational modifications. The expressedCAR can be within the cytoplasm of a host cell, into the extracellularmilieu such as the growth medium of a cell culture or anchored to thecell membrane.

As used herein, the terms “peptide,” “polypeptide,” or “protein” canrefer to a molecule comprised of amino acids and can be recognized as aprotein by those of skill in the art. The conventional one-letter orthree-letter code for amino acid residues is used herein. The terms“peptide,” “polypeptide,” and “protein” can be used interchangeablyherein to refer to polymers of amino acids of any length. The polymercan be linear or branched, it can comprise modified amino acids, and itcan be interrupted by non-amino acids. The terms also encompass an aminoacid polymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art.

The peptide sequences described herein are written according to theusual convention whereby the N-terminal region of the peptide is on theleft and the C-terminal region is on the right. Although isomeric formsof the amino acids are known, it is the L-form of the amino acid that isrepresented unless otherwise expressly indicated.

As used herein, the term “engineered immune cell” refers to an immunecell, also referred to as an immune effector cell, that has beengenetically modified by the addition of exogenous genetic material inthe form of DNA or RNA to the total genetic material of the cell.

As used herein, a “porcine tesehovirus-1 2A peptide” or “P2A peptide” or“P2A”, refers to a “self-cleaving peptide” of a picornavirus. Theaverage length of P2A peptides is 18-22 amino acids. A P2A peptide wasfirst identified in a foot-and-mouth disease virus (FMDV), a member ofthe picornavirus (Ryan et al., J Gen Virol, 1991, 72(Pt 11): 2727-2732).It was reported that ribosomes skip the synthesis of the glycyl-prolylpeptide bond at the C-terminus of a 2A peptide, leading to the cleavagebetween a 2A peptide and its immediate downstream peptide (see, e.g.,Donnelly et al., J Gen Virol., 2001, 82: 1013-1025. An exemplary P2Apeptide useful for the application comprises an amino acid sequence atleast 90%, such as 90%, 91%, 92%, 93%, 04%, 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 73. In some embodiment, the P2A peptideuseful for the application comprises the amino acid sequence of SEQ IDNO: 73.

Induced Pluripotent Stem Cells (IPSCs) and Immune Effector Cells

IPSCs have unlimited self-renewing capacity. Use of iPSCs enablescellular engineering to produce a controlled cell bank of modified cellsthat can be expanded and differentiated into desired immune effectorcells, supplying large amounts of homogeneous allogeneic therapeuticproducts.

Provided herein are genetically engineered IPSCs and derivative cellsthereof. The selected genomic modifications provided herein enhance thetherapeutic properties of the derivative cells. The derivative cells arefunctionally improved and suitable for allogenic off-the-shelf celltherapies following a combination of selective modalities beingintroduced to the cells at the level of iPSC through genomicengineering. This approach can help to reduce the side effects mediatedby CRS/GVHD and prevent long-term autoimmunity while providing excellentefficacy.

As used herein, the term “differentiation” is the process by which anunspecialized (“uncommitted”) or less specialized cell acquires thefeatures of a specialized cell. Specialized cells include, for example,a blood cell or a muscle cell. A differentiated ordifferentiation-induced cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term“committed”, when applied to the process of differentiation, refers to acell that has proceeded in the differentiation pathway to a point where,under normal circumstances, it will continue to differentiate into aspecific cell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. As used herein, the term “pluripotent”refers to the ability of a cell to form all lineages of the body or somaor the embryo proper. For example, embryonic stem cells are a type ofpluripotent stem cells that are able to form cells from each of thethree germs layers, the ectoderm, the mesoderm, and the endoderm.Pluripotency is a continuum of developmental potencies ranging from theincompletely or partially pluripotent cell (e.g., an epiblast stem cellor EpiSC), which is unable to give rise to a complete organism to themore primitive, more pluripotent cell, which is able to give rise to acomplete organism (e.g., an embryonic stem cell).

As used herein, the terms “reprogramming” or “dedifferentiation” refersto a method of increasing the potency of a cell or dedifferentiating thecell to a less differentiated state. For example, a cell that has anincreased cell potency has more developmental plasticity (i.e., candifferentiate into more cell types) compared to the same cell in thenon-reprogrammed state. In other words, a reprogrammed cell is one thatis in a less differentiated state than the same cell in anon-reprogrammed state.

As used herein, the term “induced pluripotent stem cells” or, iPSCs,means that the stem cells are produced from differentiated adult,neonatal or fetal cells that have been induced or changed orreprogrammed into cells capable of differentiating into tissues of allthree germ or dermal layers: mesoderm, endoderm, and ectoderm. The iPSCsproduced do not refer to cells as they are found in nature.

The term “hematopoietic stem and progenitor cells,” “hematopoietic stemcells,” “hematopoietic progenitor cells,” or “hematopoietic precursorcells” or “HPCs” refers to cells which are committed to a hematopoieticlineage but are capable of further hematopoietic differentiation.Hematopoietic stem cells include, for example, multipotent hematopoieticstem cells (hematoblasts), myeloid progenitors, megakaryocyteprogenitors, erythrocyte progenitors, and lymphoid progenitors.Hematopoietic stem and progenitor cells (HSCs) are multipotent stemcells that give rise to all the blood cell types including myeloid(monocytes and macrophages, neutrophils, basophils, eosinophils,erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoidlineages (T cells, B cells, NK cells). As used herein, “CD34+hematopoietic progenitor cell” refers to an HPC that expresses CD34 onits surface.

As used herein, the term “immune cell” or “immune effector cell” refersto a cell that is involved in an immune response. Immune responseincludes, for example, the promotion of an immune effector response.Examples of immune cells include T cells, B cells, natural killer (NK)cells, mast cells, and myeloid-derived phagocytes.

As used herein, the terms “T lymphocyte” and “T cell” are usedinterchangeably and refer to a type of white blood cell that completesmaturation in the thymus and that has various roles in the immunesystem. A T cell can have the roles including, e.g., the identificationof specific foreign antigens in the body and the activation anddeactivation of other immune cells. A T cell can be any T cell, such asa cultured T cell, e.g., a primary T cell, or a T cell from a cultured Tcell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from amammal. The T cell can be CD3+ cells. The T cell can be any type of Tcell and can be of any developmental stage, including but not limitedto, CD4+/CD8+ double positive T cells, CD4+ helper T cells (e.g., Thland Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral bloodmononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumorinfiltrating lymphocytes (TILs), memory T cells, naive T cells,regulator T cells, gamma delta T cells (gd T cells), and the like.Additional types of helper T cells include cells such as Th3 (Treg),Thl7, Th9, or Tfh cells. Additional types of memory T cells includecells such as central memory T cells (Tcm cells), effector memory Tcells (Tern cells and TEMRA cells). The T cell can also refer to agenetically engineered T cell, such as a T cell modified to express a Tcell receptor (TCR) or a chimeric antigen receptor (CAR). The T cell canalso be differentiated from a stem cell or progenitor cell.

“CD4+ T cells” refers to a subset of T cells that express CD4 on theirsurface and are associated with cell-mediated immune response. They arecharacterized by the secretion profiles following stimulation, which mayinclude secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4and IL10. “CD4” are 55-kD glycoproteins originally defined asdifferentiation antigens on T-lymphocytes, but also found on other cellsincluding monocytes/macrophages. CD4 antigens are members of theimmunoglobulin supergene family and are implicated as associativerecognition elements in MHC (major histocompatibility complex) classII-restricted immune responses. On T-lymphocytes they define thehelper/inducer subset.

“CD8+ T cells” refers to a subset of T cells which express CD8 on theirsurface, are MHC class I-restricted, and function as cytotoxic T cells.“CD8” molecules are differentiation antigens found on thymocytes and oncytotoxic and suppressor T-lymphocytes. CD8 antigens are members of theimmunoglobulin supergene family and are associative recognition elementsin major histocompatibility complex class I-restricted interactions.

As used herein, the term “NK cell” or “Natural Killer cell” refers to asubset of peripheral blood lymphocytes defined by the expression of CD56and CD45 and the absence of the T cell receptor (TCR chains). The NKcell can also refer to a genetically engineered NK cell, such as a NKcell modified to express a chimeric antigen receptor (CAR). The NK cellcan also be differentiated from a stem cell or progenitor cell.

As used herein, the term “genetic imprint” refers to genetic orepigenetic information that contributes to preferential therapeuticattributes in a source cell or an iPSC, and is retainable in the sourcecell derived iPSCs, and/or the iPSC-derived hematopoietic lineage cells.As used herein, “a source cell” is a non-pluripotent cell that may beused for generating iPSCs through reprogramming, and the source cellderived iPSCs may be further differentiated to specific cell typesincluding any hematopoietic lineage cells. The source cell derivediPSCs, and differentiated cells therefrom are sometimes collectivelycalled “derived” or “derivative” cells depending on the context. Forexample, derivative effector cells, or derivative NK or “iNK” cells orderivative T or “iT” cells, as used throughout this application arecells differentiated from an iPSC, as compared to their primarycounterpart obtained from natural/native sources such as peripheralblood, umbilical cord blood, or other donor tissues. As used herein, thegenetic imprint(s) conferring a preferential therapeutic attribute isincorporated into the iPSCs either through reprogramming a selectedsource cell that is donor-, disease-, or treatment response-specific, orthrough introducing genetically modified modalities to iPSC usinggenomic editing.

The induced pluripotent stem cell (iPSC) parental cell lines may begenerated from peripheral blood mononuclear cells (PBMCs) or T-cellsusing any known method for introducing re-programming factors intonon-pluripotent cells such as the episomal plasmid-based process aspreviously described in U.S. Pat. Nos. 8,546,140; 9,644,184; 9,328,332;and 8,765,470, the complete disclosures of which are incorporated hereinby reference. The reprogramming factors may be in a form ofpolynucleotides, and thus are introduced to the non-pluripotent cells byvectors such as a retrovirus, a Sendai virus, an adenovirus, an episome,and a mini-circle. In particular embodiments, the one or morepolynucleotides encoding at least one reprogramming factor areintroduced by a lentiviral vector. In some embodiments, the one or morepolynucleotides introduced by an episomal vector. In various otherembodiments, the one or more polynucleotides are introduced by a Sendaiviral vector. In some embodiments, the iPSC's are clonal iPSC's or areobtained from a pool of iPSCs and the genome edits are introduced bymaking one or more targeted integration and/or in/del at one or moreselected sites. In another embodiment, the iPSC's are obtained fromhuman T cells having antigen specificity and a reconstituted TCR gene(hereinafter, also refer to as “T-iPS” cells) as described in US Pat.Nos. 9206394, and 10787642 hereby incorporated by reference into thepresent application.

According to a particular aspect, the application relates to an inducedpluripotent stem cell (iPSC) cell or a derivative cell thereofcomprising: (i) a first exogenous polynucleotide encoding a chimericantigen receptor (CAR); (ii) a second exogenous polynucleotide encodinga truncated epithelial growth factor receptor (tEGFR) variant and aninterleukin 15 (IL-15), wherein the tEGFR variant and IL-15 are operablylinked by an autoprotease peptide, such as a porcine tesehovirus-1 2A(P2A) peptide; and (iii) a deletion or reduced expression of B2M andCIITA genes.

I. Chimeric Antigen Receptor (CAR) Expression

According to embodiments of the application, an iPSC cell or aderivative cell thereof comprises a first exogenous polynucleotideencoding a chimeric antigen receptor (CAR), such as a CAR targeting atumor antigen. In one embodiment, the CAR targets a CD19 antigen.

As used herein, the term “chimeric antigen receptor” (CAR) refers to arecombinant polypeptide comprising at least an extracellular domain thatbinds specifically to an antigen or a target, a transmembrane domain andan intracellular signaling domain. Engagement of the extracellulardomain of the CAR with the target antigen on the surface of a targetcell results in clustering of the CAR and delivers an activationstimulus to the CAR-containing cell. CARs redirect the specificity ofimmune effector cells and trigger proliferation, cytokine production,phagocytosis and/or production of molecules that can mediate cell deathof the target antigen-expressing cell in a major histocompatibility(MHC)-independent manner.

As used herein, the term “signal peptide” refers to a leader sequence atthe amino-terminus (N-terminus) of a nascent CAR protein, whichco-translationally or post-translationally directs the nascent proteinto the endoplasmic reticulum and subsequent surface expression.

As used herein, the term “extracellular antigen binding domain,”“extracellular domain,” or “extracellular ligand binding domain” refersto the part of a CAR that is located outside of the cell membrane and iscapable of binding to an antigen, target or ligand.

As used herein, the term “hinge region” or “hinge domain” refers to thepart of a CAR that connects two adjacent domains of the CAR protein,i.e., the extracellular domain and the transmembrane domain of the CARprotein.

As used herein, the term “transmembrane domain” refers to the portion ofa CAR that extends across the cell membrane and anchors the CAR to cellmembrane.

As used herein, the term “intracellular signaling domain,” “cytoplasmicsignaling domain,” or “intracellular signaling domain” refers to thepart of a CAR that is located inside of the cell membrane and is capableof transducing an effector signal.

As used herein, the term “stimulatory molecule” refers to a moleculeexpressed by an immune cell (e.g., NK cell or T cell) that provides theprimary cytoplasmic signaling sequence(s) that regulate primaryactivation of receptors in a stimulatory way for at least some aspect ofthe immune cell signaling pathway. Stimulatory molecules comprise twodistinct classes of cytoplasmic signaling sequence, those that initiateantigen-dependent primary activation (referred to as “primary signalingdomains”), and those that act in an antigen-independent manner toprovide a secondary of co-stimulatory signal (referred to as“co-stimulatory signaling domains”).

In certain embodiments, the extracellular domain comprises an antigenbinding domain and/or an antigen binding fragment. The antigen bindingfragment can, for example, be an antibody or antigen binding fragmentthereof that specifically binds a tumor antigen. The antigen bindingfragments of the application possess one or more desirable functionalproperties, including but not limited to high-affinity binding to atumor antigen, high specificity to a tumor antigen, the ability tostimulate complement-dependent cytotoxicity (CDC), antibody-dependentphagocytosis (ADPC), and/or antibody-dependent cellular-mediatedcytotoxicity (ADCC) against cells expressing a tumor antigen, and theability to inhibit tumor growth in subjects in need thereof and inanimal models when administered alone or in combination with otheranti-cancer therapies.

As used herein, the term “antibody” is used in a broad sense andincludes immunoglobulin or antibody molecules including human,humanized, composite and chimeric antibodies and antibody fragments thatare monoclonal or polyclonal. In general, antibodies are proteins orpeptide chains that exhibit binding specificity to a specific antigen.Antibody structures are well known. Immunoglobulins can be assigned tofive major classes (i.e., IgA, IgD, IgE, IgG and IgM), depending on theheavy chain constant domain amino acid sequence. IgA and IgG are furthersub-classified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.Accordingly, the antibodies of the application can be of any of the fivemajor classes or corresponding sub-classes. Preferably, the antibodiesof the application are IgG1, IgG2, IgG3 or IgG4. Antibody light chainsof vertebrate species can be assigned to one of two clearly distincttypes, namely kappa and lambda, based on the amino acid sequences oftheir constant domains. Accordingly, the antibodies of the applicationcan contain a kappa or lambda light chain constant domain. According toparticular embodiments, the antibodies of the application include heavyand/or light chain constant regions from rat or human antibodies. Inaddition to the heavy and light constant domains, antibodies contain anantigen-binding region that is made up of a light chain variable regionand a heavy chain variable region, each of which contains three domains(i.e., complementarity determining regions 1-3; CDR1, CDR2, and CDR3).The light chain variable region domains are alternatively referred to asLCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains arealternatively referred to as HCDR1, HCDR2, and HCDR3.

As used herein, the term an “isolated antibody” refers to an antibodywhich is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds to the specific tumor antigen is substantially free of antibodiesthat do not bind to the tumor antigen). In addition, an isolatedantibody is substantially free of other cellular material and/orchemicals.

As used herein, the term “monoclonal antibody” refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that can be present inminor amounts. The monoclonal antibodies of the application can be madeby the hybridoma method, phage display technology, single lymphocytegene cloning technology, or by recombinant DNA methods. For example, themonoclonal antibodies can be produced by a hybridoma which includes a Bcell obtained from a transgenic nonhuman animal, such as a transgenicmouse or rat, having a genome comprising a human heavy chain transgeneand a light chain transgene.

As used herein, the term “antigen-binding fragment” refers to anantibody fragment such as, for example, a diabody, a Fab, a Fab′, aF(ab′)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a(dsFv)₂, a bispecific dsFv (dsFv-dsFv′), a disulfide stabilized diabody(ds diabody), a single-chain antibody molecule (scFv), a single domainantibody (sdAb), a scFv dimer (bivalent diabody), a multispecificantibody formed from a portion of an antibody comprising one or moreCDRs, a camelized single domain antibody, a minibody, a nanobody, adomain antibody, a bivalent domain antibody, a light chain variabledomain (VL), a variable domain (V_(H)H) of a camelid antibody, or anyother antibody fragment that binds to an antigen but does not comprise acomplete antibody structure. An antigen-binding fragment is capable ofbinding to the same antigen to which the parent antibody or a parentantibody fragment binds.

As used herein, the term “single-chain antibody” refers to aconventional single-chain antibody in the field, which comprises a heavychain variable region and a light chain variable region connected by ashort peptide of about 15 to about 20 amino acids (e.g., a linkerpeptide).

As used herein, the term “single domain antibody” refers to aconventional single domain antibody in the field, which comprises aheavy chain variable region and a heavy chain constant region or whichcomprises only a heavy chain variable region.

As used herein, the term “human antibody” refers to an antibody producedby a human or an antibody having an amino acid sequence corresponding toan antibody produced by a human made using any technique known in theart. This definition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide.

As used herein, the term “humanized antibody” refers to a non-humanantibody that is modified to increase the sequence homology to that of ahuman antibody, such that the antigen-binding properties of the antibodyare retained, but its antigenicity in the human body is reduced.

As used herein, the term “chimeric antibody” refers to an antibodywherein the amino acid sequence of the immunoglobulin molecule isderived from two or more species. The variable region of both the lightand heavy chains often corresponds to the variable region of an antibodyderived from one species of mammal (e.g., mouse, rat, rabbit, etc.)having the desired specificity, affinity, and capability, while theconstant regions correspond to the sequences of an antibody derived fromanother species of mammal (e.g., human) to avoid eliciting an immuneresponse in that species.

As used herein, the term “multispecific antibody” refers to an antibodythat comprises a plurality of immunoglobulin variable domain sequences,wherein a first immunoglobulin variable domain sequence of the pluralityhas binding specificity for a first epitope and a second immunoglobulinvariable domain sequence of the plurality has binding specificity for asecond epitope. In an embodiment, the first and second epitopes are onthe same antigen, e.g., the same protein (or subunit of a multimericprotein). In an embodiment, the first and second epitopes overlap orsubstantially overlap. In an embodiment, the first and second epitopesdo not overlap or do not substantially overlap. In an embodiment, thefirst and second epitopes are on different antigens, e.g., the differentproteins (or different subunits of a multimeric protein). In anembodiment, a multispecific antibody comprises a third, fourth, or fifthimmunoglobulin variable domain. In an embodiment, a multi specificantibody is a bispecific antibody molecule, a trispecific antibodymolecule, or a tetraspecific antibody molecule.

As used herein, the term “bispecific antibody” refers to a multispecificantibody that binds no more than two epitopes or two antigens. Abispecific antibody is characterized by a first immunoglobulin variabledomain sequence which has binding specificity for a first epitope and asecond immunoglobulin variable domain sequence that has bindingspecificity for a second epitope. In an embodiment, the first and secondepitopes are on the same antigen, e.g., the same protein (or subunit ofa multimeric protein). In an embodiment, the first and second epitopesoverlap or substantially overlap. In an embodiment, the first and secondepitopes are on different antigens, e.g., the different proteins (ordifferent subunits of a multimeric protein). In an embodiment, abispecific antibody comprises a heavy chain variable domain sequence anda light chain variable domain sequence which have binding specificityfor a first epitope and a heavy chain variable domain sequence and alight chain variable domain sequence which have binding specificity fora second epitope. In an embodiment, a bispecific antibody comprises ahalf antibody, or fragment thereof, having binding specificity for afirst epitope and a half antibody, or fragment thereof, having bindingspecificity for a second epitope. In an embodiment, a bispecificantibody comprises a scFv, or fragment thereof, having bindingspecificity for a first epitope, and a scFv, or fragment thereof, havingbinding specificity for a second epitope. In an embodiment, a bispecificantibody comprises a V_(H)H having binding specificity for a firstepitope, and a V_(H)H having binding specificity for a second epitope.

As used herein, an antigen binding domain or antigen binding fragmentthat “specifically binds to a tumor antigen” refers to an antigenbinding domain or antigen binding fragment that binds a tumor antigen,with a KD of 1×10⁻⁷ M or less, preferably 1×10⁻⁸ M or less, morepreferably 5×10⁻⁹M or less, 1×10⁻⁹M or less, 5×10⁻¹⁰ M or less, or1×10⁻¹⁰ M or less. The term “KD” refers to the dissociation constant,which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and isexpressed as a molar concentration (M). KD values for antibodies can bedetermined using methods in the art in view of the present disclosure.For example, the KD of an antigen binding domain or antigen bindingfragment can be determined by using surface plasmon resonance, such asby using a biosensor system, e.g., a Biacore® system, or by usingbio-layer interferometry technology, such as an Octet RED96 system.

The smaller the value of the KD of an antigen binding domain or antigenbinding fragment, the higher affinity that the antigen binding domain orantigen binding fragment binds to a target antigen.

In various embodiments, antibodies or antibody fragments suitable foruse in the CAR of the present disclosure include, but are not limitedto, monoclonal antibodies, bispecific antibodies, multispecificantibodies, chimeric antibodies, polypeptide-Fc fusions, single-chainFvs (scFv), single chain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked Fvs (sdFv), masked antibodies (e.g., Probodies®), SmallModular ImmunoPharmaceuticals (“SMIPs™”), intrabodies, minibodies,single domain antibody variable domains, nanobodies, VHHs, diabodies,tandem diabodies (TandAb®), anti-idiotypic (anti-Id) antibodies(including, e.g., anti-Id antibodies to antigen-specific TCR), andepitope-binding fragments of any of the above. Antibodies and/orantibody fragments may be derived from murine antibodies, rabbitantibodies, human antibodies, fully humanized antibodies, camelidantibody variable domains and humanized versions, shark antibodyvariable domains and humanized versions, and camelized antibody variabledomains.

In some embodiments, the antigen-binding fragment is an Fab fragment, anFab′ fragment, an F(ab′)2 fragment, an scFv fragment, an Fv fragment, adsFv diabody, a VHH, a VNAR, a single-domain antibody (sdAb) ornanobody, a dAb fragment, a Fd′ fragment, a Fd fragment, a heavy chainvariable region, an isolated complementarity determining region (CDR), adiabody, a triabody, or a decabody. In some embodiments, theantigen-binding fragment is an scFv fragment. In some embodiments, theantigen-binding fragment is a VHH.

In some embodiments, at least one of the extracellular tag-bindingdomain, the antigen-binding domain, or the tag comprises a single-domainantibody or nanobody.

In some embodiments, at least one of the extracellular tag-bindingdomain, the antigen-binding domain, or the tag comprises a VHH.

In some embodiments, the extracellular tag-binding domain and the tageach comprise a VHH.

In some embodiments, the extracellular tag-binding domain, the tag, andthe antigen-binding domain each comprise a VHH.

In some embodiments, at least one of the extracellular tag-bindingdomain, the antigen-binding domain, or the tag comprises an scFv.

In some embodiments, the extracellular tag-binding domain and the tageach comprise an scFv.

In some embodiments, the extracellular tag-binding domain, the tag, andthe antigen-binding domain each comprise a scFv.

Alternative scaffolds to immunoglobulin domains that exhibit similarfunctional characteristics, such as high-affinity and specific bindingof target biomolecules, may also be used in the CARs of the presentdisclosure. Such scaffolds have been shown to yield molecules withimproved characteristics, such as greater stability or reducedimmunogenicity. Non-limiting examples of alternative scaffolds that maybe used in the CAR of the present disclosure include engineered,tenascin-derived, tenascin type III domain (e.g., Centyrin™);engineered, gamma-B crystallin-derived scaffold or engineered,ubiquitin-derived scaffold (e.g., Affilins); engineered,fibronectin-derived, 10th fibronectin type III (10Fn3) domain (e.g.,monobodies, AdNectins™ or AdNexins™); engineered, ankyrin repeat motifcontaining polypeptide (e.g., DARPins™); engineered,low-density-lipoprotein-receptor-derived, A domain (LDLR-A) (e.g.,Avimers™); lipocalin (e.g., anticalins); engineered, proteaseinhibitor-derived, Kunitz domain (e.g., EETI-II/AGRP,BPTI/LACI-D1/ITI-D2); engineered, Protein-A-derived, Z domain(Affibodies™); Sac7d-derived polypeptides (e.g., Nanoffitins® oraffitins); engineered, Fyn-derived, SH2 domain (e.g., Fynomers®); CTLD₃(e.g., Tetranectin); thioredoxin (e.g., peptide aptamer); KALBITOR®; theβ-sandwich (e.g., iMab); miniproteins; C-type lectin-like domainscaffolds; engineered antibody mimics; and any genetically manipulatedcounterparts of the foregoing that retains its binding functionality(Wörn A, Pluckthun A, J Mol Biol 305: 989-1010 (2001); Xu L et al., ChemBiol 9: 933-42 (2002); Wikman M et al., Protein Eng Des Sel 17: 455-62(2004); Binz H et al., Nat Biolechnol 23: 1257-68 (2005); Hey T et al.,Trends Biotechnol 23:514-522 (2005); Holliger P, Hudson P, NatBiotechnol 23: 1126-36 (2005); Gill D, Damle N, Curr Opin Biotech 17:653-8 (2006); Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007);Skerra, Current Opin. in Biotech., 2007 18: 295-304; Byla P et al., JBiol Chem 285: 12096 (2010); Zoller F et al., Molecules 16: 2467-85(2011), each of which is incorporated by reference in its entirety).

In some embodiments, the alternative scaffold is Affilin or Centyrin.

In some embodiments, the first polypeptide of the CARs of the presentdisclosure comprises a leader sequence. The leader sequence may bepositioned at the N-terminus the extracellular tag-binding domain. Theleader sequence may be optionally cleaved from the extracellulartag-binding domain during cellular processing and localization of theCAR to the cellular membrane. Any of various leader sequences known toone of skill in the art may be used as the leader sequence. Non-limitingexamples of peptides from which the leader sequence may be derivedinclude granulocyte-macrophage colony-stimulating factor receptor(GMCSFR), FcεR, human immunoglobulin (IgG) heavy chain (HC) variableregion, CD8α, or any of various other proteins secreted by T cells. Invarious embodiments, the leader sequence is compatible with thesecretory pathway of a T cell. In certain embodiments, the leadersequence is derived from human immunoglobulin heavy chain (HC).

In some embodiments, the leader sequence is derived from GMCSFR. In oneembodiment, the GMCSFR leader sequence comprises the amino acid sequenceset forth in SEQ ID NO: 1, or a variant thereof having at least 50, atleast 55, at least 60, at least 65, at least 70, at least 75, at least80, at least 85, at least 90, at least 95, at least 96, at least 97, atleast 98 or at least 99%, sequence identity with SEQ ID NO: 1.

In some embodiments, the first polypeptide of the CARs of the presentdisclosure comprise a transmembrane domain, fused in frame between theextracellular tag-binding domain and the cytoplasmic domain.

The transmembrane domain may be derived from the protein contributing tothe extracellular tag-binding domain, the protein contributing thesignaling or co-signaling domain, or by a totally different protein. Insome instances, the transmembrane domain can be selected or modified byamino acid substitution, deletions, or insertions to minimizeinteractions with other members of the CAR complex. In some instances,the transmembrane domain can be selected or modified by amino acidsubstitution, deletions, or insertions to avoid binding of proteinsnaturally associated with the transmembrane domain. In certainembodiments, the transmembrane domain includes additional amino acids toallow for flexibility and/or optimal distance between the domainsconnected to the transmembrane domain.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Non-limiting examplesof transmembrane domains of particular use in this disclosure may bederived from (i.e. comprise at least the transmembrane region(s) of) theα, β or ζ chain of the T-cell receptor (TCR), CD28, CD3 epsilon, CD45,CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80,CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domainmay be synthetic, in which case it will comprise predominantlyhydrophobic residues such as leucine and valine. For example, a tripletof phenylalanine, tryptophan and/or valine can be found at each end of asynthetic transmembrane domain.

In some embodiments, it will be desirable to utilize the transmembranedomain of the ζ, η or FcεR1γ chains which contain a cysteine residuecapable of disulfide bonding, so that the resulting chimeric proteinwill be able to form disulfide linked dimers with itself, or withunmodified versions of the ζ, η or FcεR1γ chains or related proteins. Insome instances, the transmembrane domain will be selected or modified byamino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex. Inother cases, it will be desirable to employ the transmembrane domain ofζ, η or FcεR1γ and −β, MB1 (Igα), B29 or CD3-γ, ζ, or η, in order toretain physical association with other members of the receptor complex.

In some embodiments, the transmembrane domain is derived from CD8 orCD28. In one embodiment, the CD8 transmembrane domain comprises theamino acid sequence set forth in SEQ ID NO: 23, or a variant thereofhaving at least 50, at least 55, at least 60, at least 65, at least 70,at least 75, at least 80, at least 85, at least 90, at least 95, atleast 96, at least 97, at least 98 or at least 99%, sequence identitywith SEQ ID NO: 23. In one embodiment, the CD28 transmembrane domaincomprises the amino acid sequence set forth in SEQ ID NO: 24, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 24.

In some embodiments, the first polypeptide of the CAR of the presentdisclosure comprises a spacer region between the extracellulartag-binding domain and the transmembrane domain, wherein the tag-bindingdomain, linker, and the transmembrane domain are in frame with eachother.

The term “spacer region” as used herein generally means any oligo- orpolypeptide that functions to link the tag-binding domain to thetransmembrane domain. A spacer region can be used to provide moreflexibility and accessibility for the tag-binding domain. A spacerregion may comprise up to 300 amino acids, preferably 10 to 100 aminoacids and most preferably 25 to 50 amino acids. A spacer region may bederived from all or part of naturally occurring molecules, such as fromall or part of the extracellular region of CD8, CD4 or CD28, or from allor part of an antibody constant region. Alternatively, the spacer regionmay be a synthetic sequence that corresponds to a naturally occurringspacer region sequence, or may be an entirely synthetic spacer regionsequence. Non-limiting examples of spacer regions which may be used inaccordance to the disclosure include a part of human CD8a chain, partialextracellular domain of CD28, FcyRllla receptor, IgG, IgM, IgA, IgD,IgE, an Ig hinge, or functional fragment thereof. In some embodiments,additional linking amino acids are added to the spacer region to ensurethat the antigen-binding domain is an optimal distance from thetransmembrane domain. In some embodiments, when the spacer is derivedfrom an Ig, the spacer may be mutated to prevent Fc receptor binding.

In some embodiments, the spacer region comprises a hinge domain. Thehinge domain may be derived from CD8α, CD28, or an immunoglobulin (IgG).For example, the IgG hinge may be from IgG1, IgG2, IgG3, IgG4, IgM1,IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof.

In certain embodiments, the hinge domain comprises an immunoglobulin IgGhinge or functional fragment thereof. In certain embodiments, the IgGhinge is from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE,or a chimera thereof. In certain embodiments, the hinge domain comprisesthe CH1, CH2, CH3 and/or hinge region of the immunoglobulin. In certainembodiments, the hinge domain comprises the core hinge region of theimmunoglobulin. The term “core hinge” can be used interchangeably withthe term “short hinge” (a.k.a “SH”). Non-limiting examples of suitablehinge domains are the core immunoglobulin hinge regions includeEPKSCDKTHTCPPCP (SEQ ID NO: 57) from IgG1, ERKCCVECPPCP (SEQ ID NO: 58)from IgG2, ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)₃ (SEQ ID NO: 59) fromIgG3, and ESKYGPPCPSCP (SEQ ID NO: 60) from IgG4 (see also Wypych etal., JBC 2008 283(23): 16194-16205, which is incorporated herein byreference in its entirety for all purposes). In certain embodiments, thehinge domain is a fragment of the immunoglobulin hinge.

In some embodiments, the hinge domain is derived from CD8 or CD28. Inone embodiment, the CD8 hinge domain comprises the amino acid sequenceset forth in SEQ ID NO: 21, or a variant thereof having at least 50, atleast 55, at least 60, at least 65, at least 70, at least 75, at least80, at least 85, at least 90, at least 95, at least 96, at least 97, atleast 98 or at least 99%, sequence identity with SEQ ID NO: 21. In oneembodiment, the CD28 hinge domain comprises the amino acid sequence setforth in SEQ ID NO: 22, or a variant thereof having at least 50, atleast 55, at least 60, at least 65, at least 70, at least 75, at least80, at least 85, at least 90, at least 95, at least 96, at least 97, atleast 98 or at least 99%, sequence identity with SEQ ID NO: 22.

In some embodiments, the transmembrane domain and/or hinge domain isderived from CD8 or CD28. In some embodiments, both the transmembranedomain and hinge domain are derived from CD8. In some embodiments, boththe transmembrane domain and hinge domain are derived from CD28.

In certain aspects, the first polypeptide of CARs of the presentdisclosure comprise a cytoplasmic domain, which comprises at least oneintracellular signaling domain. In some embodiments, cytoplasmic domainalso comprises one or more co-stimulatory signaling domains.

The cytoplasmic domain is responsible for activation of at least one ofthe normal effector functions of the host cell (e.g., T cell) in whichthe CAR has been placed in. The term “effector function” refers to aspecialized function of a cell. Effector function of a T-cell, forexample, may be cytolytic activity or helper activity including thesecretion of cytokines. Thus, the term “signaling domain” refers to theportion of a protein which transduces the effector function signal anddirects the cell to perform a specialized function. While usually theentire signaling domain is present, in many cases it is not necessary touse the entire chain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact chain as long as it transduces the effectorfunction signal. The term intracellular signaling domain is thus meantto include any truncated portion of the signaling domain sufficient totransduce the effector function signal.

Non-limiting examples of signaling domains which can be used in the CARsof the present disclosure include, e.g., signaling domains derived fromDAP10, DAP12, Fc epsilon receptor I γ chain (FCER1G), FcR β, CD3δ, CD3ε,CD3γ, CD3ζ, CD5, CD22, CD226, CD66d, CD79A, and CD79B.

In some embodiments, the cytoplasmic domain comprises a CD3ζ signalingdomain. In one embodiment, the CD3ζ signaling domain comprises the aminoacid sequence set forth in SEQ ID NO: 6, or a variant thereof having atleast 50, at least 55, at least 60, at least 65, at least 70, at least75, at least 80, at least 85, at least 90, at least 95, at least 96, atleast 97, at least 98 or at least 99%, sequence identity with SEQ ID NO:6.

In some embodiments, the cytoplasmic domain further comprises one ormore co-stimulatory signaling domains. In some embodiments, the one ormore co-stimulatory signaling domains are derived from CD28, 41BB,IL2Rb, CD40, OX40 (CD134), CD80, CD86, CD27, ICOS, NKG2D, DAP10, DAP12,2B4 (CD244), BTLA, CD30, GITR, CD226, CD79A, and HVEM.

In one embodiment, the co-stimulatory signaling domain is derived from41BB. In one embodiment, the 41BB co-stimulatory signaling domaincomprises the amino acid sequence set forth in SEQ ID NO: 8, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 8.

In one embodiment, the co-stimulatory signaling domain is derived fromIL2Rb. In one embodiment, the IL2Rb co-stimulatory signaling domaincomprises the amino acid sequence set forth in SEQ ID NO: 9, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 9.

In one embodiment, the co-stimulatory signaling domain is derived fromCD40. In one embodiment, the CD40 co-stimulatory signaling domaincomprises the amino acid sequence set forth in SEQ ID NO: 10, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 10.

In one embodiment, the co-stimulatory signaling domain is derived fromOX40. In one embodiment, the OX40 co-stimulatory signaling domaincomprises the amino acid sequence set forth in SEQ ID NO: 11, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 11.

In one embodiment, the co-stimulatory signaling domain is derived fromCD80. In one embodiment, the CD80 co-stimulatory signaling domaincomprises the amino acid sequence set forth in SEQ ID NO: 12, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 12.

In one embodiment, the co-stimulatory signaling domain is derived fromCD86. In one embodiment, the CD86 co-stimulatory signaling domaincomprises the amino acid sequence set forth in SEQ ID NO: 13, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 13.

In one embodiment, the co-stimulatory signaling domain is derived fromCD27. In one embodiment, the CD27 co-stimulatory signaling domaincomprises the amino acid sequence set forth in SEQ ID NO: 14, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 14.

In one embodiment, the co-stimulatory signaling domain is derived fromICOS. In one embodiment, the ICOS co-stimulatory signaling domaincomprises the amino acid sequence set forth in SEQ ID NO: 15, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 15.

In one embodiment, the co-stimulatory signaling domain is derived fromNKG2D. In one embodiment, the NKG2D co-stimulatory signaling domaincomprises the amino acid sequence set forth in SEQ ID NO: 16, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 16.

In one embodiment, the co-stimulatory signaling domain is derived fromDAP10. In one embodiment, the DAP10 co-stimulatory signaling domaincomprises the amino acid sequence set forth in SEQ ID NO: 17, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 17.

In one embodiment, the co-stimulatory signaling domain is derived fromDAP12. In one embodiment, the DAP12 co-stimulatory signaling domaincomprises the amino acid sequence set forth in SEQ ID NO: 18, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 18.

In one embodiment, the co-stimulatory signaling domain is derived from2B4 (CD244). In one embodiment, the 2B4 (CD244) co-stimulatory signalingdomain comprises the amino acid sequence set forth in SEQ ID NO: 19, ora variant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 19.

In some embodiments, the CAR of the present disclosure comprises onecostimulatory signaling domains. In some embodiments, the CAR of thepresent disclosure comprises two or more costimulatory signalingdomains. In certain embodiments, the CAR of the present disclosurecomprises two, three, four, five, six or more costimulatory signalingdomains.

In some embodiments, the signaling domain(s) and costimulatory signalingdomain(s) can be placed in any order. In some embodiments, the signalingdomain is upstream of the costimulatory signaling domains. In someembodiments, the signaling domain is downstream from the costimulatorysignaling domains. In the cases where two or more costimulatory domainsare included, the order of the costimulatory signaling domains could beswitched.

Non-limiting exemplary CAR regions and sequences are provided in Table1.

TABLE 1 CAR SEQ ID regions Sequence UniProt Id NO CD19 CAR: GMCSFRMLLLVTSLLLCELPHPAFLLIP 1 Signal Peptide FMC63 VHEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG 2 VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY YYGGSYAMDYWGQGTSVTVSS WhitlowGSTSGSGKPGSGEGSTKG 3 Linker FMC63 VL DIQMTQTTSSLSASLGDRVTISCRASQDISKYLN4 WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGS GTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT CD28 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFP P10747-1 5 (AA 114-220)GPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD3-zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEY P20963-3 6 isoform 3DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ (AA 52-163)KDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR FMC63 scFVEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG 7 VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKH YYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCR ASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ GNTLPYTFGGGTKLEIT Signaling Domains:41BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP Q07011 8 (AA 214-255) EEEEGGCELIL2Rb NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHG P14784 9 (AA 266-551)GDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERD KVTQLLPLNTDAYLSLQELQGQDPTHLV CD40KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTA P25942 10 (AA 216-277)APVQETLHGCQPVTQEDGKESRISVQERQ OX40 ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQAP43489 11 (AA 236-277) DAHSTLAKI CD80 TYCFAPRCRERRRNERLRRESVRPV P3368112 (AA 264-288) CD86 KWKKKKRPRNSYKCGTNTMEREESEQTKKRE P42081 13(AA269-329) KIHIPERSDEAQRVFKSSKTSSCDKSDTCF CD27QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIP P26842 14 (AA 213-260) IQEDYRKPEPACSPICOS CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKS Q9Y6W8 15 (AA 162-199) RLTDVTLNKG2D MGWIRGRRSR HSWEMSEFHN YNLDLKKSDF P26718 16 (AA 1-51)STRWQKQRCPVVKSKCRENAS DAP10 LCARPRRSPAQEDGKVYINMPGRG Q9UBK5 17(AA 70-93) DAP12 YFLGRLVPRGRGAAEAATRKQRITETESPYQEL O54885 18 (AA 62-113)QGQRSDVYSDLNTQRPYYK 2B4/CD244 WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRN Q9BZW8 19(AA 251-370) HEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQP KAQNPARLSRKELENFDVYS CD3-zetaRVKFSRSADAPAYQQGQNQLYNELNLGRREEY P20963-3 6 isoform 3DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ (AA 52-163)KDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR CD28RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP P10747-1 20 (AA 180-220) PRDFAAYRSSpacer/Hinge: CD8 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA P01732 21(AA 136-182) VHTRGLDFACDIY CD28 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPP10747-1 22 (AA 114-151) GPSKP Transmembrane: CD8 IYIWAPLAGTCGVLLLSLVITP01732 23 (AA 183-203) CD28 FWVLVVVGGVLACYSLLVTVAFIIFWV P10747-1 24(AA 153-179) Linkers: Whitlow GSTSGSGKPGSGEGSTKG 3 Linker (G₄S)₃GGGGSGGGGSGGGGS 25 Linker 3 GGSEGKSSGSGSESKSTGGS 26 Linker 4 GGGSGGGS 27Linker 5 GGGSGGGSGGGS 28 Linker 6 GGGSGGGSGGGSGGGS 29 Linker 7GGGSGGGSGGGSGGGSGGGS 30 Linker 8 GGGGSGGGGSGGGGSGGGGS 31 Linker 9GGGGSGGGGSGGGGSGGGGSGGGGS 32 Linker 10 IRPRAIGGSKPRVA 33 Linker 11GKGGSGKGGSGKGGS 34 Linker 12 GGKGSGGKGSGGKGS 35 Linker 13GGGKSGGGKSGGGKS 36 Linker 14 GKGKSGKGKSGKGKS 37 Linker 15GGGKSGGKGSGKGGS 38 Linker 16 GKPGSGKPGSGKPGS 39 Linker 17GKPGSGKPGSGKPGSGKPGS 40 Linker 18 GKGKSGKGKSGKGKSGKGKS 41 Linker 19STAGDTHLGGEDFD 42 Linker 20 GEGGSGEGGSGEGGS 43 Linker 21 GGEGSGGEGSGGEGS44 Linker 22 GEGESGEGESGEGES 45 Linker 23 GGGESGGEGSGEGGS 46 Linker 24GEGESGEGESGEGESGEGES 47 Linker 25 GSTSGSGKPGSGEGSTKG 48 Linker 26PRGASKSGSASQTGSAPGS 49 Linker 27 GTAAAGAGAAGGAAAGAAG 50 Linker 28GTSGSSGSGSGGSGSGGGG 51 Linker 29 GKPGSGKPGSGKPGSGKPGS 52 Linker 30 GSGS53 Linker 31 APAPAPAPAP 54 Linker 32 APAPAPAPAPAPAPAPAPAP 55 Linker 33AEAAAKEAAAKEAAAAKEAAAAKEAAAAKAAA 56

In some embodiments, the antigen-binding domain of the secondpolypeptide binds to an antigen. The antigen-binding domain of thesecond polypeptide may bind to more than one antigen or more than oneepitope in an antigen. For example, the antigen-binding domain of thesecond polypeptide may bind to two, three, four, five, six, seven, eightor more antigens. As another example, the antigen-binding domain of thesecond polypeptide may bind to two, three, four, five, six, seven, eightor more epitopes in the same antigen.

The choice of antigen-binding domain may depend upon the type and numberof antigens that define the surface of a target cell. For example, theantigen-binding domain may be chosen to recognize an antigen that actsas a cell surface marker on target cells associated with a particulardisease state. In certain embodiments, the CARs of the presentdisclosure can be genetically modified to target a tumor antigen ofinterest by way of engineering a desired antigen-binding domain thatspecifically binds to an antigen (e.g., on a tumor cell). Non-limitingexamples of cell surface markers that may act as targets for theantigen-binding domain in the CAR of the disclosure include thoseassociated with tumor cells or autoimmune diseases.

In some embodiments, the antigen-binding domain binds to at least onetumor antigen or autoimmune antigen.

In some embodiments, the antigen-binding domain binds to at least onetumor antigen. In some embodiments, the antigen-binding domain binds totwo or more tumor antigens. In some embodiments, the two or more tumorantigens are associated with the same tumor. In some embodiments, thetwo or more tumor antigens are associated with different tumors.

In some embodiments, the antigen-binding domain binds to at least oneautoimmune antigen. In some embodiments, the antigen-binding domainbinds to two or more autoimmune antigens. In some embodiments, the twoor more autoimmune antigens are associated with the same autoimmunedisease. In some embodiments, the two or more autoimmune antigens areassociated with different autoimmune diseases.

In some embodiments, the tumor antigen is associated with glioblastoma,ovarian cancer, cervical cancer, head and neck cancer, liver cancer,prostate cancer, pancreatic cancer, renal cell carcinoma, bladdercancer, or hematologic malignancy. Non-limiting examples of tumorantigen associated with glioblastoma include HER2, EGFRvIII, EGFR,CD133, PDGFRA, FGFR1, FGFR3, MET, CD70, ROBO1 and IL13Rα2. Non-limitingexamples of tumor antigens associated with ovarian cancer include FOLR1,FSHR, MUC16, MUC1, Mesothelin, CA125, EpCAM, EGFR, PDGFRα, Nectin-4, andB7H4. Non-limiting examples of the tumor antigens associated withcervical cancer or head and neck cancer include GD2, MUC1, Mesothelin,HER2, and EGFR. Non-limiting examples of tumor antigen associated withliver cancer include Claudin 18.2, GPC-3, EpCAM, cMET, and AFP.Non-limiting examples of tumor antigens associated with hematologicalmalignancies include CD22, CD79, BCMA, GPRC5D, SLAM F7, CD33, CLL1,CD123, and CD70. Non-limiting examples of tumor antigens associated withbladder cancer include Nectin-4 and SLITRK6.

Additional examples of antigens that may be targeted by theantigen-binding domain include, but are not limited to,alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba 733,BrE3-antigen, carbonic anhydrase EX, CD1, CD1a, CD3, CD5, CD15, CD16,CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a,CD80, CD123, CD138, colon-specific antigen-p (CSAp), CEA (CEACAM5),CEACAM6, CSAp, EGFR, EGP-I, EGP-2, Ep-CAM, EphA1, EphA2, EphA3, EphA4,EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6,FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin(HCG) and its subunits, hypoxia inducible factor (HIF-I), Ia, IL-2,IL-6, IL-8, insulin growth factor-1 (IGF-I), KC4-antigen, KS-1-antigen,KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC2, MUC3, MUC4,NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placentalgrowth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, S100,TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreichantigens, tumor necrosis antigens, VEGF, ED-B fibronectin,17-1A-antigen, an angiogenesis marker, an oncogene marker or an oncogeneproduct.

In one embodiment, the antigen targeted by the antigen-binding domain isCD19. In one embodiment, the antigen-binding domain comprises ananti-CD19 scFv. In one embodiment, the anti-CD19 scFv comprises a heavychain variable region (VH) comprising the amino acid sequence set forthin SEQ ID NO: 2, or a variant thereof having at least 50, at least 55,at least 60, at least 65, at least 70, at least 75, at least 80, atleast 85, at least 90, at least 95, at least 96, at least 97, at least98 or at least 99%, sequence identity with SEQ ID NO: 2. In oneembodiment, the anti-CD19 scFv comprises a light chain variable region(VL) comprising the amino acid sequence set forth in SEQ ID NO: 4, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 4. In one embodiment, the anti-CD19scFv comprises the amino acid sequence set forth in SEQ ID NO: 7, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 7.

In some embodiments, the antigen is associated with an autoimmunedisease or disorder. Such antigens may be derived from cell receptorsand cells which produce “self”-directed antibodies. In some embodiments,the antigen is associated with an autoimmune disease or disorder such asRheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome,Systemic lupus erythematosus, sarcoidosis, Type 1 diabetes mellitus,insulin dependent diabetes mellitus (IDDM), autoimmune thyroiditis,reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, chronicinflammatory demyelinating polyneuropathy, Guillain-Barre syndrome,Crohn's disease or ulcerative colitis.

In some embodiments, autoimmune antigens that may be targeted by the CARdisclosed herein include but are not limited to platelet antigens,myelin protein antigen, Sm antigens in snRNPs, islet cell antigen,Rheumatoid factor, and anticitrullinated protein. citrullinated proteinsand peptides such as CCP-1, CCP-2 (cyclical citrullinated peptides),fibrinogen, fibrin, vimentin, filaggrin, collagen I and II peptides,alpha-enolase, translation initiation factor 4G1, perinuclear factor,keratin, Sa (cytoskeletal protein vimentin), components of articularcartilage such as collagen II, IX, and XI, circulating serum proteinssuch as RFs (IgG, IgM), fibrinogen, plasminogen, ferritin, nuclearcomponents such as RA33/hnRNP A2, Sm, eukaryotic translation elongationfactor 1 alpha 1, stress proteins such as HSP-65, -70, -90, BiP,inflammatory/immune factors such as B7-H1, IL-1 alpha, and IL-8, enzymessuch as calpastatin, alpha-enolase, aldolase-A, dipeptidyl peptidase,osteopontin, glucose-6-phosphate isomerase, receptors such as lipocortin1, neutrophil nuclear proteins such as lactoferrin and 25-35 kD nuclearprotein, granular proteins such as bactericidal permeability increasingprotein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3,platelet antigens, myelin protein antigen, islet cell antigen,rheumatoid factor, histones, ribosomal P proteins, cardiolipin,vimentin, nucleic acids such as dsDNA, ssDNA, and RNA, ribonuclearparticles and proteins such as Sm antigens (including but not limited toSmD's and SmB′/B), U1RNP, A2/B1 hnRNP, Ro (SSA), and La (SSB) antigens.

In various embodiments, the scFv fragment used in the CAR of the presentdisclosure may include a linker between the VH and VL domains. Thelinker can be a peptide linker and may include any naturally occurringamino acid. Exemplary amino acids that may be included into the linkerare Gly, Ser Pro, Thr, Glu, Lys, Arg, Ile, Leu, His and The. The linkershould have a length that is adequate to link the VH and the VL in sucha way that they form the correct conformation relative to one another sothat they retain the desired activity, such as binding to an antigen.The linker may be about 5-50 amino acids long. In some embodiments, thelinker is about 10-40 amino acids long. In some embodiments, the linkeris about 10-35 amino acids long. In some embodiments, the linker isabout 10-30 amino acids long. In some embodiments, the linker is about10-25 amino acids long. In some embodiments, the linker is about 10-20amino acids long. In some embodiments, the linker is about 15-20 aminoacids long. Exemplary linkers that may be used are Gly rich linkers, Glyand Ser containing linkers, Gly and Ala containing linkers, Ala and Sercontaining linkers, and other flexible linkers.

In one embodiment, the linker is a Whitlow linker. In one embodiment,the Whitlow linker comprises the amino acid sequence set forth in SEQ IDNO: 3, or a variant thereof having at least 50, at least 55, at least60, at least 65, at least 70, at least 75, at least 80, at least 85, atleast 90, at least 95, at least 96, at least 97, at least 98 or at least99%, sequence identity with SEQ ID NO: 3. In another embodiment, thelinker is a (G₄S)₃ linker. In one embodiment, the (G₄S)₃ linkercomprises the amino acid sequence set forth in SEQ ID NO: 25, or avariant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 25. Other linker sequences may includeportions of immunoglobulin hinge area, CL or CH1 derived from anyimmunoglobulin heavy or light chain isotype. Exemplary linkers that maybe used include any of SEQ ID NOs: 26-56 in Table 1. Additional linkersare described for example in Int. Pat. Publ. No. WO2019/060695,incorporated by reference herein in its entirety.

II. Artificial Cell Death Polypeptide

According to embodiments of the application, an iPSC cell or aderivative cell thereof comprises a second exogenous polynucleotideencoding an artificial cell death polypeptide.

As used herein, the term “artificial cell death polypeptide” refers toan engineered protein designed to prevent potential toxicity orotherwise adverse effects of a cell therapy. The artificial cell deathpolypeptide could mediate induction of apoptosis, inhibition of proteinsynthesis, DNA replication, growth arrest, transcriptional andpost-transcriptional genetic regulation and/or antibody-mediateddepletion. In some instance, the artificial cell death polypeptide isactivated by an exogenous molecule, e.g. an antibody, that whenactivated, triggers apoptosis and/or cell death of a therapeutic cell.

In certain embodiments, an artificial cell death polypeptide comprisesan inactivated cell surface receptor that comprises an epitopespecifically recognized by an antibody, particularly a monoclonalantibody, which is also referred to herein as a monoclonalantibody-specific epitope. When expressed by iPSCs or derivative cellsthereof, the inactivated cell surface receptor is signaling inactive orsignificantly impaired, but can still be specifically recognized by anantibody. The specific binding of the antibody to the inactivated cellsurface receptor enables the elimination of the iPSCs or derivativecells thereof by ADCC and/or ADCP mechanisms, as well as, direct killingwith antibody drug conjugates with toxins or radionuclides.

In certain embodiments, the inactivated cell surface receptor comprisesan epitope that is selected from epitopes specifically recognized by anantibody, including but not limited to, ibritumomab, tiuxetan,muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin,cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumabpegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab,omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab,trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab,golimumab, ipilimumab, ofatumumab, panitumumab, or ustekinumab.

Epidermal growth factor receptor, also known as EGFR, ErbB1 and HER1, isa cell-surface receptor for members of the epidermal growth factorfamily of extracellular ligands. As used herein, “truncated EGFR,”“tEGFR,” “short EGFR” or “sEGFR” refers to an inactive EGFR variant thatlacks the EGF-binding domains and the intracellular signaling domains ofthe EGFR. An exemplary tEGFR variant contains residues 322-333 of domain2, all of domains 3 and 4 and the transmembrane domain of the nativeEGFR sequence containing the cetuximab binding epitope. Expression ofthe tEGFR variant on the cell surface enables cell elimination by anantibody that specifically binds to the tEGFR, such as cetuximab(Erbitux®), as needed. Due to the absence of the EGF-binding domains andintracellular signaling domains, tEGFR is inactive when expressed byiPSCs or derivative cell thereof.

An exemplary inactivated cell surface receptor of the applicationcomprises a tEGFR variant. In certain embodiments, expression of theinactivated cell surface receptor in an engineered immune cellexpressing a chimeric antigen receptor (CAR) induces cell suicide of theengineered immune cell when the cell is contacted with an anti-EGFRantibody. Methods of using inactivated cell surface receptors aredescribed in WO2019/070856, WO2019/023396, WO2018/058002, the disclosureof which is incorporated herein by reference. For example, a subject whohas previously received an engineered immune cell of the presentdisclosure that comprises a heterologous polynucleotide encoding aninactivated cell surface receptor comprising a tEGFR variant can beadministered an anti-EGFR antibody in an amount effective to ablate inthe subject the previously administered engineered immune cell.

In certain embodiments, the anti-EGFR antibody is cetuximab, matuzumab,necitumumab or panitumumab, preferably the anti-EGFR antibody iscetuximab.

In certain embodiments, the tEGFR variant comprises or consists of anamino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 71,preferably the amino acid sequence of SEQ ID NO: 71.

In some embodiments, the inactivated cell surface receptor comprises oneor more epitopes of CD79b, such as an epitope specifically recognized bypolatuzumab vedotin. In certain embodiments, the CD79b epitope comprisesor consists of an amino acid sequence at least 90%, such as at least90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical toSEQ ID NO: 78, preferably the amino acid sequence of SEQ ID NO: 78.

In some embodiments, the inactivated cell surface receptor comprises oneor more epitopes of CD20, such as an epitope specifically recognized byrituximab. In certain embodiments, the CD20 epitope comprises orconsists of an amino acid sequence at least 90%, such as at least 90%,91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ IDNO: 80, preferably the amino acid sequence of SEQ ID NO: 80.

In some embodiments, the inactivated cell surface receptor comprises oneor more epitopes of Her 2 receptor or ErbB, such as an epitopespecifically recognized by trastuzumab. In certain embodiments, themonoclonal antibody-specific epitope comprises or consists of an aminoacid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 82, preferablythe amino acid sequence of SEQ ID NO: 82.

In some embodiments the inactivated cell surface receptor furthercomprises a cytokine, such as interleukin-15 or interleukin-2.

As used herein “Interleukin-15” or “IL-15” refers to a cytokine thatregulates T and NK cell activation and proliferation, or a functionalportion thereof. A “functional portion” (“biologically active portion”)of a cytokine refers to a portion of the cytokine that retains one ormore functions of full length or mature cytokine. Such functions forIL-15 include the promotion of NK cell survival, regulation of NK celland T cell activation and proliferation as well as the support of NKcell development from hematopoietic stem cells. As will be appreciatedby those of skill in the art, the sequence of a variety of IL-15molecules are known in the art. In certain embodiments, the IL-15 is awild-type IL-15. In certain embodiments, the IL-15 is a human IL-15. Incertain embodiments, the IL-15 comprises an amino acid sequence at least90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%, identical to SEQ ID NO: 72, preferably the amino acid sequenceof SEQ ID NO: 72.

As used herein “Interleukin-2” refers to a cytokine that regulates T andNK cell activation and proliferation, or a functional portion thereof.In certain embodiments, the IL-2 is a wild-type IL-2. In certainembodiments, the IL-2 is a human IL-2. In certain embodiments, the IL-2comprises an amino acid sequence at least 90%, such as at least 90%,91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ IDNO: 76, preferably the amino acid sequence of SEQ ID NO: 76.

In certain embodiments, an inactivated cell surface receptor comprises amonoclonal antibody-specific epitope operably linked to a cytokine,preferably by an autoprotease peptide. Examples of the autoproteasepeptide include, but are not limited to, a peptide sequence selectedfrom the group consisting of porcine teschovirus-1 2A (P2A), afoot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus(ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmicpolyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), and acombination thereof. In one embodiment, the autoprotease peptidecomprises or is an autoprotease peptide of a porcine tesehovirus-1 2A(P2A) peptide. In certain embodiments, the autoprotease peptidecomprises an amino acid sequence at least 90%, such as at least 90%,91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ IDNO: 73, preferably the amino acid sequence of SEQ ID NO: 73.

In certain embodiments, an inactivated cell surface receptor comprises atruncated epithelial growth factor receptor (tEGFR) variant operablylinked to an interleukin-15 (IL-15) or IL-2 by an autoprotease peptide.In a particular embodiment, the inactivated cell surface receptorcomprises an amino acid sequence at least 90%, such as at least 90%,91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ IDNO: 74, preferably the amino acid sequence of SEQ ID NO: 74.

In some embodiments, an inactivated cell surface receptor furthercomprises a signal sequence. In certain embodiments, the signal sequencecomprises an amino acid sequence at least 90%, such as at least 90%,91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ IDNO: 77, preferably the amino acid sequence of SEQ ID NO: 77.

In some embodiments, an inactivated cell surface receptor furthercomprises a hinge domain. In some embodiments, the hinge domain isderived from CD8. In one embodiment, the CD8 hinge domain comprises theamino acid sequence set forth in SEQ ID NO: 21, or a variant thereofhaving at least 50, at least 55, at least 60, at least 65, at least 70,at least 75, at least 80, at least 85, at least 90, at least 95, atleast 96, at least 97, at least 98 or at least 99%, sequence identitywith SEQ ID NO: 21.

In certain embodiments, an inactivated cell surface receptor furthercomprises a transmembrane domain. In some embodiments, the transmembranedomain is derived from CD8. In one embodiment, the CD8 transmembranedomain comprises the amino acid sequence set forth in SEQ ID NO: 23, ora variant thereof having at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, at least 96, at least 97, at least 98 or at least 99%,sequence identity with SEQ ID NO: 23.

In certain embodiment, an inactivated cell surface receptor comprisesone or more epitopes specifically recognized by an antibody in itsextracellular domain, a transmembrane region and a cytoplasmic domain.In some embodiments, the inactivated cell surface receptor furthercomprises a hinge region between the epitope(s) and the transmembraneregion. In some embodiments, the inactivated cell surface receptorcomprises more than one epitopes specifically recognized by an antibody,the epitopes can have the same or different amino acid sequences, andthe epitopes can be linked together via a peptide linker, such as aflexible peptide linker have the sequence of (GGGGS)n, wherein n is aninteger of 1-8 (SEQ ID NO: 25). In some embodiments, the inactivatedcell surface receptor further comprises a cytokine, such as an IL-15 orIL-2. In certain embodiments, the cytokine is in the cytoplasmic domainof the inactivated cell surface receptor. Preferably, the cytokine isoperably linked to the epitope(s) specifically recognized by anantibody, directly or indirectly, via an autoprotease peptide, such asthose described herein. In some embodiments, the cytokine is indirectlylinked to the epitope(s) by connecting to the transmembrane region viathe autoprotease peptide.

Non-limiting exemplary inactivated cell surface receptor regions andsequences are provided in Table 2.

TABLE 2 SEQ ID Regions Sequence NO tEGFR-IL15: tEGFRMRPSGTAGAALLALLAALCPASRAGVRKCKKCEGPCRK 71VCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATG MVGALLLLLVVALGIGLFM P2AATNFSLLKQAGDVEENPGP 73 IL-15 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSA72 GLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS CD79b-IL15: SignalMEFGLSWVFLVALFRGVQC 77 Sequence CD79b ARSEDRYRNPKGSACSRIWQS 78 epitopeCD8 (AA TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL 21 136-182)  DFACDIYCD8 (AA IYIWAPLAGTCGVLLLSLVIT 23 183-203) P2A ATNFSLLKQAGDVEENPGP 73IL-15 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSA 72GLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS CD20 mimitope-IL15: SignalMEFGLSWVFLVALFRGVQC 77 Sequence CD20 ACPYANPSLC 80 mimitope LinkerGGGSGGGS 27 CD8 (AA TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL 21 136-182)DFACDIY CD8 (AA IYIWAPLAGTCGVLLLSLVIT 23 183-203) P2AATNFSLLKQAGDVEENPGP 73 IL-15 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSA72 GLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS ErbB epitope-IL15: SignalMEFGLSWVFLVALFRGVQC 77 Sequence ErbBEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEE 82 epitopeCRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVV GILLVVVLGVVFGILIGGGGSGG P2AATNFSLLKQAGDVEENPGP 73 IL-15 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSA72 GLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS

In a particular embodiment, the inactivated cell surface receptorcomprises an amino acid sequence at least 90%, such as at least 90%,91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ IDNO: 79, preferably the amino acid sequence of SEQ ID NO: 79.

In a particular embodiment, the inactivated cell surface receptorcomprises an amino acid sequence at least 90%, such as at least 90%,91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ IDNO: 81, preferably the amino acid sequence of SEQ ID NO: 81.

In a particular embodiment, the inactivated cell surface receptorcomprises an amino acid sequence at least 90%, such as at least 90%,91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ IDNO: 83, preferably the amino acid sequence of SEQ ID NO: 83.

III. HLA Expression

In certain embodiments, an iPSC or derivative cell thereof of theapplication can be further modified by introducing a third exogenouspolynucleotide encoding one or more proteins related to immune evasion,such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G). Inparticular, disruption of the B2M gene eliminates surface expression ofall MHC class I molecules, leaving cells vulnerable to lysis by NK cellsthrough the “missing self” response. Exogenous HLA-E expression can leadto resistance to NK-mediated lysis (Gornalusse et al., Nat Biotechnol.2017 August; 35(8): 765-772).

In certain embodiments, the iPSC or derivative cell thereof comprises athird exogenous polypeptide encoding at least one of a human leukocyteantigen E (HLA-E) and human leukocyte antigen G (HLA-G). In a particularembodiment, the HLA-E comprises an amino acid sequence at least 90%,such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100%, identical to SEQ ID NO: 65, preferably the amino acid sequence ofSEQ ID NO: 65. In a particular embodiment, the HLA-G comprises an aminoacid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 68, preferablySEQ ID NO: 68.

In certain embodiments, the third exogenous polynucleotide encodes apolypeptide comprising a signal peptide operably linked to a mature B2Mprotein that is fused to an HLA-E via a linker. In a particularembodiment, the third exogenous polypeptide comprises an amino acidsequence at least sequence at least 90%, such as at least 90%, 91%, 82%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 66.

In other embodiments, the third exogenous polynucleotide encodes apolypeptide comprising a signal peptide operably linked to a mature B2Mprotein that is fused to an HLA-G via a linker. In a particularembodiment, the third exogenous polypeptide comprises an amino acidsequence at least sequence at least 90%, such as at least 90%, 91%, 82%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 69.

IV. Other Optional Genome Edits

In one embodiment of the above described cell, the genomic editing atone or more selected sites may comprise insertions of one or moreexogenous polynucleotides encoding other additional artificial celldeath polypeptides, targeting modalities, receptors, signalingmolecules, transcription factors, pharmaceutically active proteins andpeptides, drug target candidates, or proteins promoting engraftment,trafficking, homing, viability, self-renewal, persistence, and/orsurvival of the genome-engineered iPSCs or derivative cells thereof.

In some embodiments, the exogenous polynucleotides for insertion areoperatively linked to (1) one or more exogenous promoters comprisingCMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, temporal-,tissue-, or cell type-specific promoters; or (2) one or more endogenouspromoters comprised in the selected sites comprising AAVS1, CCR5,ROSA26, collagen, HTRP, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1,or other locus meeting the criteria of a genome safe harbor. In someembodiments, the genome-engineered iPSCs generated using the abovemethod comprise one or more different exogenous polynucleotides encodingproteins comprising caspase, thymidine kinase, cytosine deaminase,B-cell CD20, ErbB2 or CD79b wherein when the genome-engineered iPSCscomprise two or more suicide genes, the suicide genes are integrated indifferent safe harbor locus comprising AAVSl, CCR5, ROSA26, collagen,HTRP, H11, H11, beta-2 microglobulin, GAPDH, TCR or RUNX1. Otherexogenous polynucleotides encoding proteins may include those encodingPET reporters, homeostatic cytokines, and inhibitory checkpointinhibitory proteins such as PD1, PD-L1, and CTLA4 as well as proteinsthat target the CD47/signal regulatory protein alpha (SIRPα) axis. Insome other embodiments, the genome-engineered iPSCs generated using themethod provided herein comprise in/del at one or more endogenous genesassociated with targeting modality, receptors, signaling molecules,transcription factors, drug target candidates, immune responseregulation and modulation, or proteins suppressing engraftment,trafficking, homing, viability, self-renewal, persistence, and/orsurvival of the iPSCs or derivative cells thereof.

V. Targeted Genome Editing at Selected Locus in iPSCs

According to embodiments of the application, one or more of theexogenous polynucleotides are integrated at one or more loci on thechromosome of an iPSC.

Genome editing, or genomic editing, or genetic editing, as usedinterchangeably herein, is a type of genetic engineering in which DNA isinserted, deleted, and/or replaced in the genome of a targeted cell.Targeted genome editing (interchangeable with “targeted genomic editing”or “targeted genetic editing”) enables insertion, deletion, and/orsubstitution at pre-selected sites in the genome. When an endogenoussequence is deleted or disrupted at the insertion site during targetedediting, an endogenous gene comprising the affected sequence can beknocked-out or knocked-down due to the sequence deletion or disruption.Therefore, targeted editing can also be used to disrupt endogenous geneexpression with precision. Similarly used herein is the term “targetedintegration,” referring to a process involving insertion of one or moreexogenous sequences at pre-selected sites in the genome, with or withoutdeletion of an endogenous sequence at the insertion site.

Targeted editing can be achieved either through a nuclease-independentapproach, or through a nuclease-dependent approach. In thenuclease-independent targeted editing approach, homologous recombinationis guided by homologous sequences flanking an exogenous polynucleotideto be inserted, through the enzymatic machinery of the host cell.

Alternatively, targeted editing could be achieved with higher frequencythrough specific introduction of double strand breaks (DSBs) by specificrare-cutting endonucleases. Such nuclease-dependent targeted editingutilizes DNA repair mechanisms including non-homologous end joining(NHEJ), which occurs in response to DSBs. Without a donor vectorcontaining exogenous genetic material, the NHEJ often leads to randominsertions or deletions (in/dels) of a small number of endogenousnucleotides. In comparison, when a donor vector containing exogenousgenetic material flanked by a pair of homology arms is present, theexogenous genetic material can be introduced into the genome duringhomology directed repair (HDR) by homologous recombination, resulting ina “targeted integration.”

Available endonucleases capable of introducing specific and targetedDSBs include, but not limited to, zinc-finger nucleases (ZFN),transcription activator-like effector nucleases (TALEN), RNA-guidedCRISPR (Clustered Regular Interspaced Short Palindromic Repeats)systems. Additionally, DICE (dual integrase cassette exchange) systemutilizing phiC31 and Bxbl integrases is also a promising tool fortargeted integration.

ZFNs are targeted nucleases comprising a nuclease fused to a zinc fingerDNA binding domain. By a “zinc finger DNA binding domain” or “ZFBD” itis meant a polypeptide domain that binds DNA in a sequence-specificmanner through one or more zinc fingers. A zinc finger is a domain ofabout 30 amino acids within the zinc finger binding domain whosestructure is stabilized through coordination of a zinc ion. Examples ofzinc fingers include, but not limited to, C2H2 zinc fingers, C3H zincfingers, and C4 zinc fingers. A “designed” zinc finger domain is adomain not occurring in nature whose design/composition resultsprincipally from rational criteria, e.g., application of substitutionrules and computerized algorithms for processing information in adatabase storing information of existing ZFP designs and binding data.See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261;see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO03/016496. A “selected” zinc finger domain is a domain not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. ZFNs aredescribed in greater detail in U.S. Pat. Nos. 7,888,121 and 7,972,854,the complete disclosures of which are incorporated herein by reference.The most recognized example of a ZFN in the art is a fusion of the Foklnuclease with a zinc finger DNA binding domain.

A TALEN is a targeted nuclease comprising a nuclease fused to a TALeffector DNA binding domain. By “transcription activator-like effectorDNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNAbinding domain” it is meant the polypeptide domain of TAL effectorproteins that is responsible for binding of the TAL effector protein toDNA. TAL effector proteins are secreted by plant pathogens of the genusXanthomonas during infection. These proteins enter the nucleus of theplant cell, bind effector-specific DNA sequences via their DNA bindingdomain, and activate gene transcription at these sequences via theirtransactivation domains. TAL effector DNA binding domain specificitydepends on an effector-variable number of imperfect 34 amino acidrepeats, which comprise polymorphisms at select repeat positions calledrepeat variable-diresidues (RVD). TALENs are described in greater detailin U.S. Patent Application No. 2011/0145940, which is hereinincorporated by reference. The most recognized example of a TALEN in theart is a fusion polypeptide of the Fold nuclease to a TAL effector DNAbinding domain.

Another example of a targeted nuclease that finds use in the subjectmethods is a targeted Spoll nuclease, a polypeptide comprising a Spol 1polypeptide having nuclease activity fused to a DNA binding domain, e.g.a zinc finger DNA binding domain, a TAL effector DNA binding domain,etc. that has specificity for a DNA sequence of interest. See, forexample, U.S. Application No. 61/555,857, the disclosure of which isincorporated herein by reference.

Additional examples of targeted nucleases suitable for the presentapplication include, but not limited to Bxbl, phiC3 1, R4, PhiBTl, andWp/SPBc/TP901-1, whether used individually or in combination.

Other non-limiting examples of targeted nucleases include naturallyoccurring and recombinant nucleases; CRISPR related nucleases fromfamilies including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, andcmr; restriction endonucleases; meganucleases; homing endonucleases, andthe like. As an example, CRISPR/Cas9 requires two major components: (1)a Cas9 endonuclease and (2) the crRNA-tracrRNA complex. Whenco-expressed, the two components form a complex that is recruited to atarget DNA sequence comprising PAM and a seeding region near PAM. ThecrRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA)to guide Cas9 to target selected sequences. These two components canthen be delivered to mammalian cells via transfection or transduction.As another example, CRISPR/Cpf1 comprises two major components: (1) aCPf1 endonuclease and (2) a crRNA. When co-expressed, the two componentsform a ribonucleoprotein (RNP) complex that is recruited to a target DNAsequence comprising PAM and a seeding region near PAM. The crRNA can becombined to form a chimeric guide RNA (gRNA) to guide Cpf1 to targetselected sequences. These two components can then be delivered tomammalian cells via transfection or transduction.

MAD7 is an engineered Cas12a variant originating from the bacteriumEubacterium rectale that has a preference for 5′-TTTN-3′ and 5′-CTTN-3′PAM sites and does not require a tracrRNA. See, for example, PCTPublication No. 2018/236548, the disclosure of which is incorporatedherein by reference.

DICE mediated insertion uses a pair of recombinases, for example, phiC31and Bxbl, to provide unidirectional integration of an exogenous DNA thatis tightly restricted to each enzymes' own small attB and attPrecognition sites. Because these target att sites are not naturallypresent in mammalian genomes, they must be first introduced into thegenome, at the desired integration site. See, for example, U.S.Application Publication No. 2015/0140665, the disclosure of which isincorporated herein by reference.

One aspect of the present application provides a construct comprisingone or more exogenous polynucleotides for targeted genome integration.In one embodiment, the construct further comprises a pair of homologousarm specific to a desired integration site, and the method of targetedintegration comprises introducing the construct to cells to enable sitespecific homologous recombination by the cell host enzymatic machinery.In another embodiment, the method of targeted integration in a cellcomprises introducing a construct comprising one or more exogenouspolynucleotides to the cell, and introducing a ZFN expression cassettecomprising a DNA-binding domain specific to a desired integration siteto the cell to enable a ZFN-mediated insertion. In yet anotherembodiment, the method of targeted integration in a cell comprisesintroducing a construct comprising one or more exogenous polynucleotidesto the cell, and introducing a TALEN expression cassette comprising aDNA-binding domain specific to a desired integration site to the cell toenable a TALEN-mediated insertion. In another embodiment, the method oftargeted integration in a cell comprises introducing a constructcomprising one or more exogenous polynucleotides to the cell,introducing a Cpf1 expression cassette, and a gRNA comprising a guidesequence specific to a desired integration site to the cell to enable aCpf1-mediated insertion. In another embodiment, the method of targetedintegration in a cell comprises introducing a construct comprising oneor more exogenous polynucleotides to the cell, introducing a Cas9expression cassette, and a gRNA comprising a guide sequence specific toa desired integration site to the cell to enable a Cas9-mediatedinsertion. In still another embodiment, the method of targetedintegration in a cell comprises introducing a construct comprising oneor more att sites of a pair of DICE recombinases to a desiredintegration site in the cell, introducing a construct comprising one ormore exogenous polynucleotides to the cell, and introducing anexpression cassette for DICE recombinases, to enable DICE-mediatedtargeted integration.

Sites for targeted integration include, but are not limited to, genomicsafe harbors, which are intragenic or extragenic regions of the humangenome that, theoretically, are able to accommodate predictableexpression of newly integrated DNA without adverse effects on the hostcell or organism. In certain embodiments, the genome safe harbor for thetargeted integration is one or more loci of genes selected from thegroup consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCRand RUNX1 genes.

In other embodiments, the site for targeted integration is selected fordeletion or reduced expression of an endogenous gene at the insertionsite. As used herein, the term “deletion” with respect to expression ofa gene refers to any genetic modification that abolishes the expressionof the gene. Examples of “deletion” of expression of a gene include,e.g., a removal or deletion of a DNA sequence of the gene, an insertionof an exogenous polynucleotide sequence at a locus of the gene, and oneor more substitutions within the gene, which abolishes the expression ofthe gene.

Genes for target deletion include, but are not limited to, genes ofmajor histocompatibility complex (MHC) class I and MHC class IIproteins. Multiple MHC class I and class II proteins must be matched forhistocompatibility in allogeneic recipients to avoid allogeneicrejection problems. “MHC deficient”, including MHC-class I deficient, orMHC-class II deficient, or both, refers to cells that either lack, or nolonger maintain, or have reduced level of surface expression of acomplete MHC complex comprising a MHC class I protein heterodimer and/ora MHC class II heterodimer, such that the diminished or reduced level isless than the level naturally detectable by other cells or by syntheticmethods. MHC class I deficiency can be achieved by functional deletionof any region of the MHC class I locus (chromosome 6p2l), or deletion orreducing the expression level of one or more MHC class-I associatedgenes including, not being limited to, beta-2 microglobulin (B2M) gene,TAP 1 gene, TAP 2 gene and Tapasin genes. For example, the B2M geneencodes a common subunit essential for cell surface expression of allMHC class I heterodimers. B2M null cells are MHC-I deficient. MHC classII deficiency can be achieved by functional deletion or reduction ofMHC-II associated genes including, not being limited to, RFXANK, CIITA,RFX5 and RFXAP. CIITA is a transcriptional coactivator, functioningthrough activation of the transcription factor RFX5 required for classII protein expression. CIITA null cells are MHC-II deficient. In certainembodiments, one or more of the exogenous polynucleotides are integratedat one or more loci of genes selected from the group consisting of B2M,TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to therebydelete or reduce the expression of the gene(s) with the integration.

In certain embodiments, the exogenous polynucleotides are integrated atone or more loci on the chromosome of the cell, preferably the one ormore loci are of genes selected from the group consisting of AAVS1,CCR5, ROSA26, collagen, HTRP, HI 1, GAPDH, RUNX1, B2M, TAPI, TAP2,Tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constantregion, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3,or TIGIT genes, provided at least one of the one or more loci is of aMHC gene, such as a gene selected from the group consisting of B2M, TAP1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes. Preferably, theone or more exogenous polynucleotides are integrated at a locus of anMHC class-I associated gene, such as a beta-2 microglobulin (B2M) gene,TAP 1 gene, TAP 2 gene or Tapasin gene; and at a locus of an MHC-IIassociated gene, such as a RFXANK, CIITA, RFX5, RFXAP, or CIITA gene;and optionally further at a locus of a safe harbor gene selected fromthe group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH,TCR and RUNX1 genes. More preferably, the one or more of the exogenouspolynucleotides are integrated at the loci of CIITA, AAVS1 and B2Mgenes.

In certain embodiments, (i) the first exogenous polynucleotide isintegrated at a locus of AAVS1 gene; (ii) the second exogenouspolypeptide is integrated at a locus of CIITA gene; and (iii) the thirdexogenous polypeptide is integrated at a locus of B2M gene; whereinintegrations of the exogenous polynucleotides delete or reduceexpression of CIITA and B2M genes.

In certain embodiments, (i) the first exogenous polynucleotide comprisesthe polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 62; (ii)the second exogenous polynucleotide comprises the polynucleotidesequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to SEQ ID NO: 75; and (iii) the thirdexogenous polynucleotide comprises the polynucleotide sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to SEQ ID NO: 67.

In certain embodiments, (i) the first exogenous polynucleotide comprisesthe polynucleotide sequence of SEQ ID NO: 62; (ii) the second exogenouspolynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75;and (iii) the third exogenous polynucleotide comprises thepolynucleotide sequence of SEQ ID NO: 67.

Derivative Cells

In another aspect, the invention relates to a cell derived fromdifferentiation of an iPSC, a derivative cell. As described above, thegenomic edits introduced into the iPSC cell are retained in thederivative cell. In certain embodiments of the derivative cell obtainedfrom iPSC differentiation, the derivative cell is a hematopoietic cell,including, but not limited to, HSCs (hematopoietic stem and progenitorcells), hematopoietic multipotent progenitor cells, T cell progenitors,NK cell progenitors, T cells, NKT cells, NK cells, B cells, antigenpresenting cells (APC), monocytes and macrophages. In certainembodiments, the derivative cell is an immune effector cell, such as aNK cell or a T cell.

In certain embodiments, the application provides a natural killer (NK)cell or a T cell comprising: (i) a first exogenous polynucleotideencoding a chimeric antigen receptor (CAR); (ii) a second exogenouspolynucleotide encoding a truncated epithelial growth factor receptor(tEGFR) variant and an interleukin 15 (IL-15), wherein the tEGFR variantand IL-15 are operably linked by an autoprotease peptide, such as anautoprotease peptide of a porcine tesehovirus-1 2A (P2A) peptide; and(iii) a deletion or reduced expression of an MHC class I associated geneand an MHC class II associated gene, such as an MHC class-I associatedgene selected from the group consisting of a B2M gene, TAP 1 gene, TAP 2gene and Tapasin gene, and an MHC-II associated gene selected from thegroup consisting of a RFXANK gene, CIITA gene, RFX5 gene, RFXAP gene,and CIITA gene, preferably the B2M gene and CIITA gene.

In certain embodiments, the NK cell or T cell further comprises a thirdexogenous polynucleotide encoding at least one of a human leukocyteantigen E (HLA-E) and a human leukocyte antigen G (HLA-G).

Also provided is a NK cell or a T cell comprising: (i) a first exogenouspolynucleotide encoding a chimeric antigen receptor (CAR) having theamino acid sequence of SEQ ID NO: 61; (ii) a second exogenouspolynucleotide encoding a truncated epithelial growth factor receptor(tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, anautoprotease peptide having the amino acid sequence of SEQ ID NO: 73,and interleukin 15 (IL-15) having the amino acid sequence of SEQ ID NO:72; and (iii) a third exogenous polynucleotide encoding a humanleukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO:66; wherein the first, second and third exogenous polynucleotides areintegrated at loci of AAVS1, CIITA and B2M genes, respectively, tothereby delete or reduce expression of CIITA and B2M.

In certain embodiments, the first exogenous polynucleotide comprises thepolynucleotide sequence of SEQ ID NO: 62; the second exogenouspolynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75;and the third exogenous polynucleotide comprises the polynucleotidesequence of SEQ ID NO: 67.

Also provided is a CD34+ hematopoietic progenitor cell (HPC) derivedfrom an induced pluripotent stem cell (iPSC) comprising: (i) a firstexogenous polynucleotide encoding a chimeric antigen receptor (CAR);(ii) a second exogenous polynucleotide encoding an inactivated cellsurface receptor that comprises a monoclonal antibody-specific epitopeand an interleukin 15 (IL-15), wherein the inactivated cell surfacereceptor and the IL-15 are operably linked by an autoprotease peptide;and (iii) a deletion or reduced expression of one or more of B2M, TAP 1,TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes.

In certain embodiments, the CD34+ HPC further comprises a thirdexogenous polynucleotide encoding a human leukocyte antigen E (HLA-E)and/or human leukocyte antigen G (HLA-G).

In certain embodiments, the CAR comprises (i) a signal peptide; (ii) anextracellular domain comprising a binding domain that specifically bindsthe CD19 antigen; (iii) a hinge region; (iv) a transmembrane domain; (v)an intracellular signaling domain; and (vi) a co-stimulatory domain,such as a co-stimulatory domain comprising a CD28 signaling domain.

Also provided is a method of manufacturing the derivative cell. Themethod comprises differentiating the iPSC under conditions for celldifferentiation to thereby obtain the derivative cell.

An iPSC of the application can be differentiated by any method known inthe art. Exemplary methods are described in U.S. Pat. Nos. 8,846,395,8,945,922, 8,318,491, WO2010/099539, WO2012/109208, WO2017/070333,WO2017/179720, WO2016/010148, WO2018/048828 and WO2019/157597, each ofwhich are herein incorporated by reference in its entirety. Thedifferentiation protocol may use feeder cells or may be feeder-free. Asused herein, “feeder cells” or “feeders” are terms describing cells ofone type that are co-cultured with cells of a second type to provide anenvironment in which the cells of the second type can grow, expand, ordifferentiate, as the feeder cells provide stimulation, growth factorsand nutrients for the support of the second cell type.

In another embodiment of the invention, the iPSC derivative cells of theinvention are NK cells which are prepared by a method of differentiatingan iPSC cell into an NK cell by subjecting the cells to adifferentiation protocol including the addition of recombinant humanIL-12p70 for the final 24 hours of culture. By including the IL-12 inthe differentiation protocol, cells that are primed with IL-12demonstrate more rapid cell killing compared to those that aredifferentiated in the absence of IL-12 (FIG. 5A). In addition, the cellsdifferentiated using the IL-12 conditions demonstrate improved cancercell growth inhibition (FIG. 5B).

Polynucleotides, Vectors, and Host Cells

(1) Nucleic Acids Encoding a CAR

In another general aspect, the invention relates to an isolated nucleicacid encoding a chimeric antigen receptor (CAR) useful for an inventionaccording to embodiments of the application. It will be appreciated bythose skilled in the art that the coding sequence of a CAR can bechanged (e.g., replaced, deleted, inserted, etc.) without changing theamino acid sequence of the protein. Accordingly, it will be understoodby those skilled in the art that nucleic acid sequences encoding CARs ofthe application can be altered without changing the amino acid sequencesof the proteins.

In certain embodiments, the isolated nucleic acid encodes a CARtargeting CD19. In a particular embodiment, the isolated nucleic acidencoding the CAR comprises a polynucleotide sequence at least 90%, suchas at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%,identical to SEQ ID NO: 62, preferably the polynucleotide sequence ofSEQ ID NO: 62.

In another general aspect, the application provides a vector comprisinga polynucleotide sequence encoding a CAR useful for an inventionaccording to embodiments of the application. Any vector known to thoseskilled in the art in view of the present disclosure can be used, suchas a plasmid, a cosmid, a phage vector or a viral vector. In someembodiments, the vector is a recombinant expression vector such as aplasmid. The vector can include any element to establish a conventionalfunction of an expression vector, for example, a promoter, ribosomebinding element, terminator, enhancer, selection marker, and origin ofreplication. The promoter can be a constitutive, inducible, orrepressible promoter. A number of expression vectors capable ofdelivering nucleic acids to a cell are known in the art and can be usedherein for production of a CAR in the cell. Conventional cloningtechniques or artificial gene synthesis can be used to generate arecombinant expression vector according to embodiments of theapplication.

In a particular aspect, the application provides vectors for targetedintegration of a CAR useful for an invention according to embodiments ofthe application. In certain embodiments, the vector comprises anexogenous polynucleotide having, in the 5′ to 3′ order, (a) a promoter;(b) a polynucleotide sequence encoding a CAR according to an embodimentof the application; and (c) a terminator/polyadenylation signal.

In certain embodiments, the promoter is a CAG promoter. In certainembodiments, the CAG promoter comprises the polynucleotide sequence atleast 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 100%, identical to SEQ ID NO: 63. Other promoters can also be used,examples of which include, but are not limited to, EF1α, UBC, CMV, SV40,PGK1, and human beta actin.

In certain embodiments, the terminator/polyadenylation signal is a SV40signal. In certain embodiments, the SV40 signal comprises thepolynucleotide sequence at least 90%, such as at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 64. Otherterminator sequences can also be used, examples of which include, butare not limited to, BGH, hGH, and PGK.

In certain embodiments, the polynucleotide sequence encoding a CARcomprises the polynucleotide sequence at least 90%, such as at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ IDNO: 62.

In some embodiment, the vector further comprises a left homology arm anda right homology arm flanking the exogenous polynucleotide. As usedherein, “left homology arm” and “right homology arm” refers to a pair ofnucleic acid sequences that flank an exogenous polynucleotide andfacilitate the integration of the exogenous polynucleotide into aspecified chromosomal locus. Sequences of the left and right armhomology arms can be designed based on the integration site of interest.In some embodiment, the left or right arm homology arm is homologous tothe left or right side sequence of the integration site.

In certain embodiments, the left homology arm comprises thepolynucleotide sequence at least 90%, such as at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 90. Incertain embodiments, the right homology arm comprises the polynucleotidesequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 100%, identical to SEQ ID NO: 91.

In a particular embodiment, the vector comprises a polynucleotidesequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO:92, preferably the polynucleotide sequence of SEQ ID NO: 92.

(2) Nucleic Acids Encoding an Inactivated Cell Surface Receptor

In another general aspect, the invention relates to an isolated nucleicacid encoding an inactivated cell surface receptor useful for aninvention according to embodiments of the application. It will beappreciated by those skilled in the art that the coding sequence of aninactivated cell surface receptor can be changed (e.g., replaced,deleted, inserted, etc.) without changing the amino acid sequence of theprotein. Accordingly, it will be understood by those skilled in the artthat nucleic acid sequences encoding an inactivated cell surfacereceptor of the application can be altered without changing the aminoacid sequences of the proteins.

In certain embodiments, an isolated nucleic acid encodes any inactivatedcell surface receptor described herein, such as that comprises amonoclonal antibody-specific epitope, and a cytokine, such as an IL-15or IL-2, wherein the monoclonal antibody-specific epitope and thecytokine are operably linked by an autoprotease peptide.

In some embodiments, the isolated nucleic acid encodes an inactivatedcell surface receptor comprising an epitope specifically recognized byan antibody, such as ibritumomab, tiuxetan, muromonab-CD3, tositumomab,abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab,rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab,eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab,palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab,vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab,ipilimumab, ofatumumab, panitumumab, or ustekinumab.

In certain embodiments, the isolated nucleic acid encodes an inactivatedcell surface receptor having a truncated epithelial growth factorreceptor (tEGFR) variant. Preferably, the inactivated cell surfacereceptor comprises an epitope specifically recognized by cetuximab,matuzumab, necitumumab or panitumumab, preferably cetuximab.

In certain embodiments, the isolated nucleic acid encodes an inactivatedcell surface receptor having one or more epitopes of CD79b, such as anepitope specifically recognized by polatuzumab vedotin.

In certain embodiments, the isolated nucleic acid encodes an inactivatedcell surface receptor having one or more epitopes of CD20, such as anepitope specifically recognized by rituximab.

In certain embodiments, the isolated nucleic acid encodes an inactivatedcell surface receptor having one or more epitopes of Her 2 receptor,such as an epitope specifically recognized by trastuzumab

In certain embodiments, the autoprotease peptide comprises or is aporcine tesehovirus-1 2A (P2A) peptide.

In certain embodiments, the truncated epithelial growth factor receptor(tEGFR) variant consists of an amino acid sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity tothe amino acid sequence of SEQ ID NO: 71.

In certain embodiments, the monoclonal antibody-specific epitopespecifically recognized by polatuzumab vedotin consists of an amino acidsequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 78.

In certain embodiments, the monoclonal antibody-specific epitopespecifically recognized by rituximab consists of an amino acid sequenceat least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100%, identical to SEQ ID NO: 80.

In certain embodiments, the monoclonal antibody-specific epitopespecifically recognized by trastuzumab consists of an amino acidsequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 82.

In certain embodiments, the IL-15 comprises an amino acid sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to the amino acid sequence of SEQ ID NO: 72.

In certain embodiments, the autoprotease peptide has an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:73.

In certain embodiments, the polynucleotide sequence encodes apolypeptide comprising an amino acid sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to theamino acid sequence of SEQ ID NO: 74.

In a particular embodiment, the isolated nucleic acid encoding theinactivated cell surface receptor comprises a polynucleotide sequence atleast 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 100%, identical to SEQ ID NO: 75, preferably the polynucleotidesequence of SEQ ID NO: 75.

In certain embodiments, the polynucleotide sequence encodes apolypeptide comprising an amino acid sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to theamino acid sequence of SEQ ID NO: 79.

In another general aspect, the application provides a vector comprisinga polynucleotide sequence encoding an inactivated cell surface receptoruseful for an invention according to embodiments of the application. Anyvector known to those skilled in the art in view of the presentdisclosure can be used, such as a plasmid, a cosmid, a phage vector or aviral vector. In some embodiments, the vector is a recombinantexpression vector such as a plasmid. The vector can include any elementto establish a conventional function of an expression vector, forexample, a promoter, ribosome binding element, terminator, enhancer,selection marker, and origin of replication. The promoter can be aconstitutive, inducible, or repressible promoter. A number of expressionvectors capable of delivering nucleic acids to a cell are known in theart and can be used herein for production of a inactivated cell surfacereceptor in the cell. Conventional cloning techniques or artificial genesynthesis can be used to generate a recombinant expression vectoraccording to embodiments of the application.

In a particular aspect, the application provides a vector for targetedintegration of an inactivated cell surface receptor useful for aninvention according to embodiments of the application. In certainembodiments, the vector comprises an exogenous polynucleotide having, inthe 5′ to 3′ order, (a) a promoter; (b) a polynucleotide sequenceencoding an inactivated cell surface receptor, such as an inactivatedcell surface receptor comprising a truncated epithelial growth factorreceptor (tEGFR) variant and an interleukin 15 (IL-15), wherein thetEGFR variant and the IL-15 are operably linked by an autoproteasepeptide, such as a porcine tesehovirus-1 2A (P2A) peptide, and (c) aterminator/polyadenylation signal.

In certain embodiments, the promoter is a CAG promoter. In certainembodiments, the CAG promoter comprises the polynucleotide sequence atleast 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 100%, identical to SEQ ID NO: 63. Other promoters can also be used,examples of which include, but are not limited to, EF1α, UBC, CMV, SV40,PGK1, and human beta actin.

In certain embodiments, the terminator/polyadenylation signal is a SV40signal. In certain embodiments, the SV40 signal comprises thepolynucleotide sequence at least 90%, such as at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 64. Otherterminator sequences can also be used, examples of which include, butare not limited to BGH, hGH, and PGK.

In certain embodiments, the polynucleotide sequence encoding aninactivated cell surface receptor comprises the polynucleotide sequenceat least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 100%, identical to SEQ ID NO: 75.

In some embodiment, the vector further comprises a left homology arm anda right homology arm flanking the exogenous polynucleotide.

In certain embodiments, the left homology arm comprises thepolynucleotide sequence at least 90%, such as at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 84. Incertain embodiments, the right homology arm comprises the polynucleotidesequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 100%, identical to SEQ ID NO: 85

In a particular embodiment, the vector comprises a polynucleotidesequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO:86, preferably the polynucleotide sequence of SEQ ID NO: 86.

(3) Nucleic Acids Encoding an HLA Construct

In another general aspect, the invention relates to an isolated nucleicacid encoding an HLA construct useful for an invention according toembodiments of the application. It will be appreciated by those skilledin the art that the coding sequence of an HLA construct can be changed(e.g., replaced, deleted, inserted, etc.) without changing the aminoacid sequence of the protein. Accordingly, it will be understood bythose skilled in the art that nucleic acid sequences encoding an HLAconstruct of the application can be altered without changing the aminoacid sequences of the proteins.

In certain embodiments, the isolated nucleic acid encodes an HLAconstruct comprising a signal peptide, such as an HLA-G signal peptide,operably linked to an HLA coding sequence, such as a coding sequence ofa mature B2M, and/or a mature HLA-E. In some embodiments, the HLA codingsequence encodes the HLA-G and B2M, which are operably linked by a4×GGGGS linker, and/or the B2M and HLA-E, which are operably linked by a3×GGGGS linker. In a particular embodiment, the isolated nucleic acidencoding the HLA construct comprises a polynucleotide sequence at least90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or100%, identical to SEQ ID NO: 67, preferably the polynucleotide sequenceof SEQ ID NO: 67. In another embodiment, the isolated nucleic acidencoding the HLA construct comprises a polynucleotide sequence at least90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or100%, identical to SEQ ID NO: 70, preferably the polynucleotide sequenceof SEQ ID NO: 70.

In another general aspect, the application provides a vector comprisinga polynucleotide sequence encoding a HLA construct useful for aninvention according to embodiments of the application. Any vector knownto those skilled in the art in view of the present disclosure can beused, such as a plasmid, a cosmid, a phage vector or a viral vector. Insome embodiments, the vector is a recombinant expression vector such asa plasmid. The vector can include any element to establish aconventional function of an expression vector, for example, a promoter,ribosome binding element, terminator, enhancer, selection marker, andorigin of replication. The promoter can be a constitutive, inducible, orrepressible promoter. A number of expression vectors capable ofdelivering nucleic acids to a cell are known in the art and can be usedherein for production of a HLA construct in the cell. Conventionalcloning techniques or artificial gene synthesis can be used to generatea recombinant expression vector according to embodiments of theapplication.

In a particular aspect, the application provides vectors for targetedintegration of a HLA construct useful for an invention according toembodiments of the application. In certain embodiments, the vectorcomprises an exogenous polynucleotide having, in the 5′ to 3′ order, (a)a promoter; (b) a polynucleotide sequence encoding an HLA construct; and(c) a terminator/polyadenylation signal.

In certain embodiments, the promoter is a CAG promoter. In certainembodiments, the CAG promoter comprises the polynucleotide sequence atleast 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 100%, identical to SEQ ID NO: 63. Other promoters can also be used,examples of which include, but are not limited to, EF1α, UBC, CMV, SV40,PGK1, and human beta actin.

In certain embodiments, the terminator/polyadenylation signal is a SV40signal. In certain embodiments, the SV40 signal comprises thepolynucleotide sequence at least 90%, such as at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 64. Otherterminator sequences can also be used, examples of which include, butare not limited to BGH, hGH, and PGK.

In certain embodiments, a polynucleotide sequence encoding a HLAconstruct comprises a signal peptide, such as a HLA-G signal peptide, amature B2M, and a mature HLA-E, wherein the HLA-G and B2M are operablylinked by a 4×GGGGS linker (SEQ ID NO: 31) and the B2M transgene andHLA-E are operably linked by a 3×GGGGS linker (SEQ ID NO: 25). Inparticular embodiments, the HLA construct comprises the polynucleotidesequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 100%, identical to SEQ ID NO: 67, preferably thepolynucleotide sequence of SEQ ID NO: 67. In another embodiment, the HLAconstruct comprises the polynucleotide sequence at least 90%, such as atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical toSEQ ID NO: 70, preferably the polynucleotide sequence of SEQ ID NO: 70.

In some embodiment, the vector further comprises a left homology arm anda right homology arm flanking the exogenous polynucleotide.

In certain embodiments, the left homology arm comprises thepolynucleotide sequence at least 90%, such as at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 87. Incertain embodiments, the right homology arm comprises the polynucleotidesequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 100%, identical to SEQ ID NO: 88.

In a particular embodiment, the vector comprises a polynucleotidesequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO:89, preferably the polynucleotide sequence of SEQ ID NO: 89.

(4) Host Cells

In another general aspect, the application provides a host cellcomprising a vector of the application and/or an isolated nucleic acidencoding a construct of the application. Any host cell known to thoseskilled in the art in view of the present disclosure can be used forrecombinant expression of exogenous polynucleotides of the application.According to particular embodiments, the recombinant expression vectoris transformed into host cells by conventional methods such as chemicaltransfection, heat shock, or electroporation, where it is stablyintegrated into the host cell genome such that the recombinant nucleicacid is effectively expressed.

Examples of host cells include, for example, recombinant cellscontaining a vector or isolated nucleic acid of the application usefulfor the production of a vector or construct of interest; or anengineered iPSC or derivative cell thereof containing one or moreisolated nucleic acids of the application, preferably integrated at oneor more chromosomal loci. A host cell of an isolated nucleic acid of theapplication can also be an immune effector cell, such as a T cell or NKcell, comprising the one or more isolated nucleic acids of theapplication. The immune effector cell can be obtained by differentiationof an engineered iPSC of the application. Any suitable method in the artcan be used for the differentiation in view of the present disclosure.The immune effector cell can also be obtained transfecting an immuneeffector cell with one or more isolated nucleic acids of theapplication.

Compositions

In another general aspect, the application provides a compositioncomprising an isolated polynucleotide of the application, a host celland/or an iPSC or derivative cell thereof of the application.

In certain embodiments, the composition further comprises one or moretherapeutic agents selected from the group consisting of a peptide, acytokine, a checkpoint inhibitor, a mitogen, a growth factor, a smallRNA, a dsRNA (double stranded RNA), siRNA, oligonucleotide, mononuclearblood cells, a vector comprising one or more polynucleic acids ofinterest, an antibody, a chemotherapeutic agent or a radioactive moiety,or an immunomodulatory drug (IMiD).

In certain embodiments, the composition is a pharmaceutical compositioncomprising an isolated polynucleotide of the application, a host celland/or an iPSC or derivative cell thereof of the application and apharmaceutically acceptable carrier. The term “pharmaceuticalcomposition” as used herein means a product comprising an isolatedpolynucleotide of the application, an isolated polypeptide of theapplication, a host cell of the application, and/or an iPSC orderivative cell thereof of the application together with apharmaceutically acceptable carrier. Polynucleotides, polypeptides, hostcells, and/or iPSCs or derivative cells thereof of the application andcompositions comprising them are also useful in the manufacture of amedicament for therapeutic applications mentioned herein.

As used herein, the term “carrier” refers to any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipidcontaining vesicle, microsphere, liposomal encapsulation, or othermaterial well known in the art for use in pharmaceutical formulations.It will be understood that the characteristics of the carrier, excipientor diluent will depend on the route of administration for a particularapplication. As used herein, the term “pharmaceutically acceptablecarrier” refers to a non-toxic material that does not interfere with theeffectiveness of a composition described herein or the biologicalactivity of a composition described herein. According to particularembodiments, in view of the present disclosure, any pharmaceuticallyacceptable carrier suitable for use in a polynucleotide, polypeptide,host cell, and/or iPSC or derivative cell thereof can be used.

The formulation of pharmaceutically active ingredients withpharmaceutically acceptable carriers is known in the art, e.g.,Remington: The Science and Practice of Pharmacy (e.g. 21st edition(2005), and any later editions). Non-limiting examples of additionalingredients include: buffers, diluents, solvents, tonicity regulatingagents, preservatives, stabilizers, and chelating agents. One or morepharmaceutically acceptable carrier may be used in formulating thepharmaceutical compositions of the application.

Methods of Use

In another general aspect, the application provides a method of treatinga disease or a condition in a subject in need thereof. The methodscomprise administering to the subject in need thereof a therapeuticallyeffective amount of cells of the application and/or a composition of theapplication. In certain embodiments, the disease or condition is cancer.The cancer can, for example, be a solid or a liquid cancer. The cancer,can, for example, be selected from the group consisting of a lungcancer, a gastric cancer, a colon cancer, a liver cancer, a renal cellcarcinoma, a bladder urothelial carcinoma, a metastatic melanoma, abreast cancer, an ovarian cancer, a cervical cancer, a head and neckcancer, a pancreatic cancer, an endometrial cancer, a prostate cancer, athyroid cancer, a glioma, a glioblastoma, and other solid tumors, and anon-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma/disease (HD), an acutelymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), achronic myelogenous leukemia (CML), a multiple myeloma (MM), an acutemyeloid leukemia (AML), and other liquid tumors. In a preferredembodiment, the cancer is a non-Hodgkin's lymphoma (NHL).

According to embodiments of the application, the composition comprises atherapeutically effective amount of an isolated polynucleotide, anisolated polypeptide, a host cell, and/or an iPSC or derivative cellthereof. As used herein, the term “therapeutically effective amount”refers to an amount of an active ingredient or component that elicitsthe desired biological or medicinal response in a subject. Atherapeutically effective amount can be determined empirically and in aroutine manner, in relation to the stated purpose.

As used herein with reference to a cell of the application and/or apharmaceutical composition of the application a therapeuticallyeffective amount means an amount of the cells and/or the pharmaceuticalcomposition that modulates an immune response in a subject in needthereof.

According to particular embodiments, a therapeutically effective amountrefers to the amount of therapy which is sufficient to achieve one, two,three, four, or more of the following effects: (i) reduce or amelioratethe severity of the disease, disorder or condition to be treated or asymptom associated therewith; (ii) reduce the duration of the disease,disorder or condition to be treated, or a symptom associated therewith;(iii) prevent the progression of the disease, disorder or condition tobe treated, or a symptom associated therewith; (iv) cause regression ofthe disease, disorder or condition to be treated, or a symptomassociated therewith; (v) prevent the development or onset of thedisease, disorder or condition to be treated, or a symptom associatedtherewith; (vi) prevent the recurrence of the disease, disorder orcondition to be treated, or a symptom associated therewith; (vii) reducehospitalization of a subject having the disease, disorder or conditionto be treated, or a symptom associated therewith; (viii) reducehospitalization length of a subject having the disease, disorder orcondition to be treated, or a symptom associated therewith; (ix)increase the survival of a subject with the disease, disorder orcondition to be treated, or a symptom associated therewith; (xi) inhibitor reduce the disease, disorder or condition to be treated, or a symptomassociated therewith in a subject; and/or (xii) enhance or improve theprophylactic or therapeutic effect(s) of another therapy.

The therapeutically effective amount or dosage can vary according tovarious factors, such as the disease, disorder or condition to betreated, the means of administration, the target site, the physiologicalstate of the subject (including, e.g., age, body weight, health),whether the subject is a human or an animal, other medicationsadministered, and whether the treatment is prophylactic or therapeutic.Treatment dosages are optimally titrated to optimize safety andefficacy.

According to particular embodiments, the compositions described hereinare formulated to be suitable for the intended route of administrationto a subject. For example, the compositions described herein can beformulated to be suitable for intravenous, subcutaneous, orintramuscular administration.

The cells of the application and/or the pharmaceutical compositions ofthe application can be administered in any convenient manner known tothose skilled in the art. For example, the cells of the application canbe administered to the subject by aerosol inhalation, injection,ingestion, transfusion, implantation, and/or transplantation. Thecompositions comprising the cells of the application can be administeredtransarterially, subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, intrapleurally, byintravenous (i.v.) injection, or intraperitoneally. In certainembodiments, the cells of the application can be administered with orwithout lymphodepletion of the subject.

The pharmaceutical compositions comprising cells of the application canbe provided in sterile liquid preparations, typically isotonic aqueoussolutions with cell suspensions, or optionally as emulsions,dispersions, or the like, which are typically buffered to a selected pH.The compositions can comprise carriers, for example, water, saline,phosphate buffered saline, and the like, suitable for the integrity andviability of the cells, and for administration of a cell composition.

Sterile injectable solutions can be prepared by incorporating cells ofthe application in a suitable amount of the appropriate solvent withvarious other ingredients, as desired. Such compositions can include apharmaceutically acceptable carrier, diluent, or excipient such assterile water, physiological saline, glucose, dextrose, or the like,that are suitable for use with a cell composition and for administrationto a subject, such as a human. Suitable buffers for providing a cellcomposition are well known in the art. Any vehicle, diluent, or additiveused is compatible with preserving the integrity and viability of thecells of the application.

The cells of the application and/or the pharmaceutical compositions ofthe application can be administered in any physiologically acceptablevehicle. A cell population comprising cells of the application cancomprise a purified population of cells. Those skilled in the art canreadily determine the cells in a cell population using various wellknown methods. The ranges in purity in cell populations comprisinggenetically modified cells of the application can be from about 50% toabout 55%, from about 55% to about 60%, from about 60% to about 65%,from about 65% to about 70%, from about 70% to about 75%, from about 75%to about 80%, from about 80% to about 85%, from about 85% to about 90%,from about 90% to about 95%, or from about 95% to about 100%. Dosagescan be readily adjusted by those skilled in the art, for example, adecrease in purity could require an increase in dosage.

The cells of the application are generally administered as a dose basedon cells per kilogram (cells/kg) of body weight of the subject to whichthe cells and/or pharmaceutical compositions comprising the cells areadministered. Generally, the cell doses are in the range of about 10⁴ toabout 10¹⁰ cells/kg of body weight, for example, about 10⁵ to about 10⁹,about 10⁵ to about 10⁸, about 10⁵ to about 10⁷, or about 10⁵ to about10⁶, depending on the mode and location of administration. In general,in the case of systemic administration, a higher dose is used than inregional administration, where the immune cells of the application areadministered in the region of a tumor and/or cancer. Exemplary doseranges include, but are not limited to, 1×10⁴ to 1×10⁸, 2×10⁴ to 1×10⁸,3×10⁴ to 1×10⁸, 4×10⁴ to 1×10⁸, 5×10⁴ to 6×10⁸, 7×10⁴ to 1×10⁸, 8×10⁴ to1×10⁸, 9×10⁴ to 1×10⁸, 1×10⁵ to 1×10⁸, 1×10⁵ to 9×10⁷, 1×10⁵ to 8×10⁷,1×10⁵ to 7×10⁷, 1×10⁵ to 6×10⁷, 1×10⁵ to 5×10⁷, 1×10⁵ to 4×10⁷, 1×10⁵ to4×10⁷, 1×10⁵ to 3×10⁷, 1×10⁵ to 2×10⁷, 1×10⁵ to 1×10⁷, 1×10⁵ to 9×10⁶,1×10⁵ to 8×10⁶, 1×10⁵ to 7×10⁶, 1×10⁵ to 6×10⁶, 1×10⁵ to 5×10⁶, 1×10⁵ to4×10⁶, 1×10⁵ to 4×10⁶, 1×10⁵ to 3×10⁶, 1×10⁵ to 2×10⁶, 1×10⁵ to 1×10⁶,2×10⁵ to 9×10⁷, 2×10⁵ to 8×10⁷, 2×10⁵ to 7×10⁷, 2×10⁵ to 6×10⁷, 2×10⁵ to5×10⁷, 2×10⁵ to 4×10⁷, 2×10⁵ to 4×10⁷, 2×10⁵ to 3×10⁷, 2×10⁵ to 2×10⁷,2×10⁵ to 1×10⁷, 2×10⁵ to 9×10⁶, 2×10⁵ to 8×10⁶, 2×10⁵ to 7×10⁶, 2×10⁵ to6×10⁶, 2×10⁵ to 5×10⁶, 2×10⁵ to 4×10⁶, 2×10⁵ to 4×10⁶, 2×10⁵ to 3×10⁶,2×10⁵ to 2×10⁶, 2×10⁵ to 1×10⁶, 3×10⁵ to 3×10⁶ cells/kg, and the like.Additionally, the dose can be adjusted to account for whether a singledose is being administered or whether multiple doses are beingadministered. The precise determination of what would be considered aneffective dose can be based on factors individual to each subject.

As used herein, the terms “treat,” “treating,” and “treatment” are allintended to refer to an amelioration or reversal of at least onemeasurable physical parameter related to a cancer, which is notnecessarily discernible in the subject, but can be discernible in thesubject. The terms “treat,” “treating,” and “treatment,” can also referto causing regression, preventing the progression, or at least slowingdown the progression of the disease, disorder, or condition. In aparticular embodiment, “treat,” “treating,” and “treatment” refer to analleviation, prevention of the development or onset, or reduction in theduration of one or more symptoms associated with the disease, disorder,or condition, such as a tumor or more preferably a cancer. In aparticular embodiment, “treat,” “treating,” and “treatment” refer toprevention of the recurrence of the disease, disorder, or condition. Ina particular embodiment, “treat,” “treating,” and “treatment” refer toan increase in the survival of a subject having the disease, disorder,or condition. In a particular embodiment, “treat,” “treating,” and“treatment” refer to elimination of the disease, disorder, or conditionin the subject.

The cells of the application and/or the pharmaceutical compositions ofthe application can be administered in combination with one or moreadditional therapeutic agents. In certain embodiments the one or moretherapeutic agents are selected from the group consisting of a peptide,a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a smallRNA, a dsRNA (double stranded RNA), siRNA, oligonucleotide, mononuclearblood cells, a vector comprising one or more polynucleic acids ofinterest, an antibody, a chemotherapeutic agent or a radioactive moiety,or an immunomodulatory drug (IMiD).

EXAMPLES

Abbreviations ABC Antibodies Bound Per Cell ADCC Antibody DependentCellular Cytotoxicity ALL Acute Lymphoblastic Leukemia ANOVA Analysis OfVariance APC Allophycocyanin ATCC American Type Culture Collection b2MBeta-2 Microglobulin BRC Baby Rabbit Complement BSA Bovine Serum AlbuminBUV Brilliant Ultra Violet BV Brilliant Violet C Celsius CAR ChimericAntigen Receptor CD Cluster Of Differentiation CDC Complement-MediatedCytotoxicity CTL Cytotoxic Lymphocyte CTV Celltrace Violet DMEMDulbecco′s Modified Eagle Medium E:T Effector To Target Ratio EC₅₀ HalfMaximal Effective Concentration EGFR Epidermal Growth Factor FACSFluorescence Activated Cell Sorting FBS Fetal Bovine Serum Fc FragmentCrystallizable FcR Fc Receptor FMO Fluorescence Minus One FSC-A ForwardScatter Area FSC-H Forward Scatter Height GRex Gas Permeable RapidExpansion MOI Multiplicity Of Infection N Number Of Animals NCI NationalCancer Institute ng Nanogram NIR Near-IR NK Natural Killer NKCM NkCulture Media NKG2A Natural Killer Group 2 Member A NLR Nuclight RedNSCLC Non-Small Cell Lung Carcinoma NSG Nod-Scid-Gamma p ProbabilityValue PBMC Peripheral Blood Mononuclear Cell PB-NK Peripheral BloodDerived Natural Killer Cell PBS Phosphate-Buffered Saline HC HemacareHLA Human Leukocyte Antigen HLA-E Human Leukocyte Antigen Class I, EHP-β-CD 2-Hydroxypropyl-Beta- Cyclodextrin hr Hour HSA Human SerumAlbumin Ig Immunoglobulin IL- Interleukin IMDM Iscove Modified DulbeccoMedia iNK Ipsc Derived Natural Killer Cell iNK Ipsc-Derived NaturalKiller IP Intraperitoneal iPSC Induced Pluripotent Stem Cell IR InfraredIU International Units IV Intravenous K Thousand kg Kilogram KO GeneticKnockout LLOD Lower Limit Of Detection mAb Monoclonal Antibody mgMicrogram mg Milligram MHC Major Histocompatibility Complex min MinutemL Milliliter mM Micromolar mm Millimeter PE Phycoerythrin perCP-Peridinin Chlorophyll Protein Cy5.5 Complex-Cyanin 5.5 pg Picograms PRR-Phycoerythrin RCU Red Calibrated Unit rhIL-2 Recombinant HumanInterleukin-2 RO Reverse Osmosis RT Room Temperature SD StandardDeviation SEM Standard Error Of The Mean SSC-A Side Scatter Area TGITumor Growth Inhibition WT Wild Type xG Times Gravity

Example 1. Cell Line Development

iPSC Development

Induced pluripotent stem cell (iPSC) parental cell lines were generatedfrom peripheral blood mononuclear cells (PBMCs) using an episomalplasmid-based process as previously described in U.S. Pat. Nos.8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosuresof which are incorporated herein by reference.

Vector (Plasmid) Production

Gene fragments (gBlocks) encoding the transgene of interest, with thepromoter, terminator, and homology arms were designed and synthesized bychemical synthesis at IDT, Inc. The gBlock gene fragments were assembledinto a pUC19 plasmid using the In-Fusion® Cloning HD Plus kit (TakaraBio; Shiga, Japan) according to manufacturer's protocol. Reactionproducts from In-Fusion Cloning, i.e. expression constructs, weretransformed into Stbl3 bacterial cells (Thermo Fisher; Waltham, Mass.)for amplification according to manufacturer's protocol. Vector (plasmid)from the amplified expression construct was purified from bacterial cellculture using the HiSpeed Plasmid Maxi Prep kit (Qiagen; Hilden,Germany) according to the manufacturer's protocol. Research gradesequencing was performed on purified plasmid DNA and evaluated byrestriction digestion to confirm transgene sequence. The concentrationof purified plasmid DNA was measured by absorbance. Additionally, theabsorbance ratio at A260/A280 nm and A260/A230 nm were measured toevaluate residual RNA and protein levels, respectively.

CITTA Targeting Plasmid

The CIITA targeting plasmid contains a CAG promoter (SEQ ID NO: 63),SV40 terminator/polyadenylation (SEQ ID NO: 64), and tEGFR-IL15 codingsequence. The tEGFR-IL15 transgene encodes tEGFR-IL15, which containsresidues 322-333 of domain 2, all of domains 3 and 4 and thetransmembrane domain of the native EGFR sequence (SEQ ID NO: 71). ThetEGFR-IL15 transgene is followed by an in frame P2A peptide sequence(SEQ ID NO: 73) and then the full-length IL-15 sequence (SEQ ID NO: 72).A schematic of the CIITA targeting transgene plasmid is shown in FIG.1A.

AA VS1 Targeting Plasmid

The AAVS1 targeting plasmid contains a CAG promoter (SEQ ID NO: 63),SV40 terminator/polyadenylation (SEQ ID NO: 64), and anti-CD19 scFvchimeric antigen receptor (CAR) sequence (SEQ ID NO: 62). The encodedCAR contains the GMCSFR signal peptide connected to the FMC63 scFvfollowed by residues 114 to 220 of CD28 and residues 52 to 163 ofCD3zeta isoform 3. A schematic of the AAVS1 targeting transgene plasmidis shown in FIG. 1B.

B2M Targeting Plasmid

The B2M targeting plasmid contains a CAG promoter (SEQ ID NO: 63), SV40terminator/polyadenylation (SEQ ID NO: 64), and Peptide-B2M-HLA-E codingsequence (SEQ ID NO: 67). The B2M-transgene encoded protein (SEQ ID NO:66) contains the signal peptide from HLA-G followed by the nine aminoacid peptide VMAPRTLIL connected to a 4×GGGGS linker, the mature B2Msequence connected to a 3×GGGGS linker and then the mature HLA-Esequence (SEQ ID NO: 65). A schematic of the B2M targeting transgeneplasmid is shown in FIG. 1C.

Transgene insertion into the B2M (exon 2) and CIITA (exon 1) results indisruption of the coding sequences and prevents translation of thefull-length sequence. Loss of expression of B2M will prevent proper MHCClass I assembly and disrupt expression. Loss of CIITA will prevent HLAII gene transcription and prevent MHC Class II expression. Insertion ofthe transgene into intron 1 of the AAVS1 locus does not result in anycoding sequence alterations. Homology arm sequences were designed to siton the 5′ and 3′ sides of a Cpf1 genome nuclease cut site and includefrom 500-1200 bp of target locus-specific sequence.

CAR-Engineered iPSC Cell Line Establishment

Cell line establishment consisted of transfection, electroporation,CAR-Engineered iPSC expansion, cell sorting, and cell cloning steps.Three serial rounds of transfection and electroporation were performedwith the associated purified plasmid DNA and recombinant Cpf1ultra/guide RNA Ribonucleoprotein (RNP) complexes that are specific to asingle target locus (either B2M, CIITA, or AAVS1). Guide RNAs (gRNAs)were selected using the Benchling™ software design tool, with alloff-target sites scoring less than 2 (0-100) (Table 3). The vastmajority of potential off-target sites were intergenic.

TABLE 3 Guide RNAs SEQ ID NO: gRNA target Sequence 93 B2MTTTACTCACGTCATCCAGCAGAGA 94 AAVS1 TTTATCTGTCCCCTCCACCCCACA 95 CIITATTTACCTTGGGGCTCTGACAGGTA

Briefly, a vial of iPSC cells from the parental cell line was thawedinto Complete Essential 8™ medium (Thermo Fisher) with H1152 Rho KinaseInhibitor, pelleted, and resuspended in Complete Essential 8 medium. Thecell suspension was then transferred to the wells of aVitronectin-coated, 6-well plate containing Complete Essential 8 mediumwith H1152 and incubated at 37° C., 5% CO₂, low O₂. Cells from one wellwere expanded into a T-75 flask. When the flask reached 60-70%confluency, it was propagated into another T-75 flask. When this flaskbecame 60-70% confluent, the cells were used for transfection. H1152 wasadded to a T-75 flask containing iPSC cells and the cells were incubatedat 37° C., 5% CO₂, low O₂.

Following incubation, cells were washed with DPBS, dissociated from theflask, and resuspended in Complete Essential 8 medium. Cells werecounted and seeded into a T-75 flask, which had been pre-coated withVitronectin, containing Complete Essential 8 medium with H1152, at anappropriate cell density for transfection. Lipofectamine Stem reagent(Thermo Fisher) and purified plasmid DNA were prepared in Opti-MEM(Thermo Fisher) and incubated. The transfection mix containing purifiedplasmid DNA was added to the cells and then incubated at 37° C., 5% CO₂,low O₂.

After transfection, cells were washed with DPBS, dissociated from theflask, and resuspended in Complete Essential 8 medium. The cells werethen washed with Opti-MEM, counted, washed with additional Opti-MEM, andresuspended in Opti-MEM at an appropriate cell density forelectroporation. Ribonucleoprotein (RNP) complex was generated bycombining Alt-R® CRISPR-Cpf1 crRNA and Alt-R® Cpf1 Ultra Nuclease (IDT;Coralville, Iowa). RNP complex and Cpf1 electroporation enhancer wereadded to the transfected cells and were electroporated. Electroporatedcells were then added to the wells of a pre-warmed, Vitronectin-coated,24-well plate containing Complete Essential 8 medium and NU7026 and wereincubated at 37° C., 5% CO₂, low O₂.

Cells were cultured for a minimum of 10 days in Complete Essential 8medium on Vitronectin-coated plates for homology directed repair tooccur. Once cells on the 24-well plate were 60-70% confluent, they weredissociated and propagated into one well of a 6-well plate. Afterreaching confluency, one well of a 6-well plate was propagated into aT-75 flask. Cells were maintained in culture for the minimum 10-dayduration, after which the culture was analyzed for the presence ofinserted transgenes and/or absence of deleted endogenous genes by flowcytometry. Cells were then subjected to flow cytometry sorting toisolate the modified population.

After each round of transfection and electroporation, the expanded,engineered cells were sorted for stable integrants by FluorescenceActivated Cell Sorting (FACS) using transgene-specific antibodies.Sorting after each round of engineering included markers from theprevious rounds and may require multiple rounds of sorting tosufficiently enrich the population for all the respective markers.Sorting was performed on a MacsQuant Tyto cell sorter (Miltenyi Biotech;Bergisch Gladbach, Germany) using fluorescently labeled antibodies tohuman HLA-E, human EGFR and a fusion protein of human CD19-Fc.

After completion of all three engineering steps and necessary rounds ofsorting, single cell clones were isolated by limited dilution cloning.Cells were washed once with DPBS and dissociated from the plate. Cellswere resuspended in Complete Essential 8 medium, filtered through a70-μm cell strainer, counted, and diluted to a final density of 1000cells/mL in Complete Essential 8 medium. The cells were then transferredin 200-μL aliquots to 9 mL StemFit® (Amsbio; Abington, United Kingdom)with 1 mL CloneR™ supplement (StemCell Technologies; Vancouver, Canada),plated at 100 μL/well, and rested for 24 hours. After resting, mediumwas changed every 48 hours until colonies were approximately 2 mm indiameter, at which time wells with single colonies were identified byvisual inspection and were split to duplicate Vitronectin-coated platescontaining Complete Essential 8 medium. Medium was changed every dayuntil cells reach greater than 50% confluency. One of the plates wasused for expanding single-cell lines into 6-well plates for use in theprocess and the other was used for characterization.

Hematopoietic Progenitor Cell (HPC) Differentiation

iPSC cells thawed from cryopreserved cell banks were grown on platescoated with Vitronectin in E8 medium supplemented with H1152 Rho KinaseInhibitor. The iPSC cells were passaged twice through dissociation withTrypLE™ (Thermo Fisher), and re-seeding on to Vitronectin plates with E8media+H1152. After two passages through dissociation TrypLE treatment,the iPSC cells were once more treated with TrypLE and the cells wereresuspended in an optimized concentration in HDM-I media plus H1152. HDMmedia contains IMDM medium, Ham's F12 medium, CTS B27 minus Vitamin Asupplement, Non Essential Amino Acids, Ascorbic Acid Mg 2-phosphate,Monothioglycerol, and Heparin. HDM-I media contains HDM+CHIR99021 GSK3inhibitor, FGF2, and VEGF. The resuspended cells were then seeded intothe appropriate vessels depending on scale. The next day (D1), 80% ofthe medium was replaced with Fresh HDM-I medium. At days 2, 3, and 4,80% of the medium was removed and replaced with HDM-II medium (HDMmedia+BMP4, FGF2, and VEGF). At day 5, HPCs may start to appear in thecultures, budding off of the cell aggregates. Once the HPCs started toappear, they were harvested 2 days later, but no earlier than day 8.Starting at day 5; every day 80% of the medium was removed, and any HPCsin the removed media are collected by centrifugation and the cellsresuspended in HDM-III (HDM+BMP4, SCF, TPO, FLT3L, and IL3) and addedback to the culture. HPCs were then harvested at day 8 or day 9(depending on day of initial appearance of the HPCs)

Natural Killer (NK) Cell and T Cell Differentiation and Activation

The HPCs were differentiated to generate NK or T cells. Cells werethawed, washed, and seeded into retronectin/DLL4-coated G-Rexbioreactors. Notch signaling, specific cytokines, and growth factorswere used for differentiation into lymphoid lineage and subsequent NK orT cell maturation and activation. During harvest, the culture wasconcentrated and washed, formulated using a defined cryopreservationmedium, and filled into AT vials using an M1 filling station. Vials werevisually inspected, cryopreserved in a controlled rate freezer, andstored in the vapor phase of a LN2 freezer.

NK and T cells can also be differentiated using feeder cells. Briefly,K562 myelogenous leukemia cells engineered to express class I molecules,CD64, 4-1BBL and transmembrane are cultured with the HCPs for asufficient time to promote differentiation of NK or T cells.

Example 2. CD19 Targeted Cytotoxicity Assay

To demonstrate CD19-specific target cell killing, cytotoxicity wasmeasured using an IncuCyte® assay (Essen Bioscience Inc.; Ann Arbor,Mich.). A CD19-knockout Reh B leukemia cell line was established. Cellswere also transduced with NucLightRed using lentivirus from EssenBiosciences (Sartorious) for use in Incucyte assay. Next, parental andCD19-knockout Reh B cell leukemia cells were co-cultured with iNK cellsexpressing FMC63 CD28z CAR (anti-CD19) at a 1:1 effector-to-target cellratio. Target cell death was measured over 72 hours. CAR iNK cellseffectively kill CD19-positive target cells (FIG. 2 ).

Example 3. CAR/IL-15 iNK Assays

In order to test the ability of iNK cells engineered to express theIL-15 transgene (CAR/IL-15 iNK) to release IL-15, CAR iNK or CAR/IL-15iNK cells were cultured in media alone or co-cultured with K562myelogenous leukemia cells (ATCC) at a 1:1 effector to target ratio.Supernatants were collected after incubating for 24, 28, 72 or 96 hoursand assayed for IL-15 concentration using an MSD immunoassay (Cat#K151URK-4) according to manufacturer's protocol (Meso Scale Diagnostic;Rockville, Md.). In both media only and with K562 targets, iNK cellsengineered to express the IL-15 transgene demonstrated superior IL-15release into the culture media (FIG. 3A).

To test the in vivo persistence of the CAR/EL-15 iNK cells, CAR iNK orCAR/IL-15 iNK cells (10E6 cells) were injected intravenously intoimmunodeficient NSG™ mice (The Jackson Laboratory; Bar Harbor, Me.) onDay 0. On Day 20 post-injection, blood and lungs were analyzed for thepresence of human CD45⁺ CD56⁺ cells (infused iNK cells) usingFluorescence-activated Cell Sorting (FACS). Only when infused cellscarried the human IL-15 transgene were they detectable after 20 days(FIG. 3B).

To further test the impact of the IL-15 transgene on the persistence ofCAG-CAR/IL-15 cells in vivo, mice were intravenously infused with 1×10⁷CAG-CAR or CAG-CAR/IL-15 cells on study Day 1. Half of the mice weresupplemented with exogenous recombinant human IL-15 (1 μg/mouse daily,intraperitoneal) for the duration of the study. On study day 8 lungswere harvested and processed for flow cytometry analysis. Samples werestained with Fixable LiveDead NearIR (Thermo fisher), anti-huCD45,anti-huCD56. During analysis iNK cells were defined as CD56/CD45 doublepositive cells and recorded as a percentage of the live cell population(FIG. 3C).

To test the ability of the CAR/IL-15 iNK cells to kill over multiplerounds of target challenge, a serial killing assay was set up with abulk culture, for repeated stimulation, in parallel to Incucyte assaysfor quantification of cytolytic activity at each round. CAR/IL-15 iNKcells were cultured at a 1:1 effector to target ratio (E:T) withIrradiated Reh cells (2Gy) for 3-4 days. Results showed thatCAR/IL-15-iNK cells perform serial killing for seven rounds beforeexhausting (FIG. 4A). At the end of the bulk culture no Reh target cellswere detectable. CAR/IL-15 iNK cells were counted on the ViCell Blue totrack expansion and allow for setting up subsequent bulk culture andIncucyte assays. Incucyte based killing assays were set up in parallelto each bulk culture, using the cells harvested from the prior bulkculture as the effector population with multiple E:Ts 5:1, 1:1, 1:5.

Next, the CAR/IL-15-iNK cells were compared to CAR-iNK cells notexpressing IL-15. The CAR/IL-15-iNK cells showed superior proliferationcompared to CAR-iNK cells (FIG. 4B). The CAR/IL-15-iNK cells also showedsuperior serial killing of Raji cells compared to CAR-iNK cells (FIG.4C).

Example 4. Cytokine Enhanced Cytotoxicity Assays

Interleukin-12 is a cytokine that stimulates the production ofinterferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from Tcells and natural killer (NK) cells. To determine whether IL-12 had aneffect on the target cytotoxicity of CAR/IL-15 iNK cells, iNK cells weredifferentiated in the standard protocol (no IL-12) or with inclusion of10 ng/ml recombinant human IL-12p70 (PeproTech; Rocky Hill, N.J.) forthe final 24 hours of culture. iNK cells were used in an Incucytekilling assay to determine efficacy for killing the Raji CD19+ B cellleukemia cell line (ATCC; Manassas, Va.). Cells that were primed withIL-12 demonstrated more rapid killing of Raji cells compared to thosethat were differentiated in the absence of IL-12 (FIG. 5A).

IL-12 primed iNK cells were further tested for effects on tumorigenesisin vivo. Luciferase-labeled Burkitt's lymphoma cell line Raji, wasintravenously (iv) implanted in female NSG™ mice on study Day 0. Micewere intravenously infused with 1×10⁷ unprimed or IL12-primedCAG-CAR-IL15 iNK cells on study days 1, 4, and 7. Mice were supplementedwith intraperitoneal recombinant human IL-2 (100,000 IU, PeproTech#200-02) three times weekly for the duration of the study, beginning onday 1. An untreated group served as the control. Mice were injected withLuciferin (VivoGlo™, Promega) prior to imaging using the IVIS SpectrumCT(Perkin Elmer). The reaction of the luciferin substrate with the fireflyluciferase enzyme produced by the Raji tumor cells, produces lightmeasured as bioluminescent signal. Data are represented as mean wholebody bioluminescent average radiance±SD. 50% and 62% tumor growthinhibition was observed with unprimed and IL-12 primed iNK treatment,respectively, at study termination on Day 20 (*p<0.05, **p<0.01) (FIG.5B).

Example 5. Elimination Assay

CAR/IL15 iNK cells were engineered to express tEGFR as an eliminationfeature, intended to operate as the target of antibody-dependentcell-mediated cytotoxicity (ADCC) and antibody-dependent cellularphagocytosis (ADCP) through dosing with Cetuximab, an EGFR inhibitor.iNK cells with or without EGFR expressed from a transgene wereco-cultured with human PBMCs. Increasing doses of Cetuximab were addedto facilitate ADCC and cells were incubated for 3 hours. Control cellswere treated with human IgG1. Results are shown in FIGS. 6A-6B. OnlyEGFR-expressing iNK cells showed dose-dependent cell death (Annexin Vstaining) in the ADCC assay. This data demonstrates that theCAR/IL15/tEGFR iNK cells can be efficiently eliminated using Cetuximab.

Example 6. MHC Class I and Class II Deletion

The plasmid constructs described in the present application are designedto target integration of exogenous polypeptides useful for the inventionof the application and simultaneously delete or reduce expression of MHCclass I and class II genes. Genomic engineering of IPSCs using the B2Mand CIITA targeting plasmids is done as described above. Afterdifferentiation to an NK cell, confirmation of MHC class I and IIexpression is confirmed using flow cytometry using antibodies specificfor HLA I (alpha chain) and HLA II (alpha or beta chain).

Example 7. Non-Classical HLA Expression

iNK cells of application are engineered to further express non-classicalHLA proteins, HLA-E or HLA-G. Expression is confirmed at all stages byflow cytometry using antibodies specific for HLA-E or HLA-G.

Example 8. iNK Mediated Lysis of K562 Cells

To demonstrate the ability iNK cells to elicit basic NK cellfunctionality, iNK clone iNK1248-iPSC611 and primary peripheral blood NK(PB-NK) cells from 3 PBMC donors were assessed for the ability to killK562 cells using the Incucyte live imaging platform. The Incucyteplatform allows for real-time quantification of fluorescently labeledtarget cells, depletion of which serves as a measurement of targetlysis.

K562 Cell Line Generation and Propagation

The chronic myelogenous leukemia (CML) cell K562 cell line was obtainedfrom ATCC. K562 cells were transduced with NucLight Red lentivirusfollowing the standard Sartorius protocol. Transduced cells wereselected and cultured in IMDM culture media containing 1 ug/mL ofpuromycin. Cells were cultured spitting every 2-3 days keeping celldensity between 1e5 cells/mL and 1e6 cells/mL.

NK Isolation

PBMCs from 3 donors were thawed in a 37 C and centrifuged for 3 min at300G. Supernatant was aspirated and cells were resuspended in RPMI+10%FBS 10 ng/mL Il-15 and rested overnight. NK cells were isolated fromrested PBMCs using CD56 MicroBeads, human (Miltenyi, part 130-050-401)according to manufacturers recommended protocol.

NK Purity Check

CAR-iNK clones and PB-NK donors were plated at 100K cells per well in 96well U-bottom plate (BD falcon 353077). All wash steps were carried outby centrifugation at 300×G for 3 min and flicking supernatants into thesink. Cells were washed 2× in PBS and stained with 100 ul of a 1:1000dilution (in PBS) of LIVE/DEAD™ Fixable Near-IR viability dye (thermoFisher) for 15 min at room temperature (RT). Cells were washed 2× in BDFACS stain buffer BSA (BD). TrustainFcX, human Fc receptor block, wasdiluted 1:100 in BD FACS stain and 50 ul of dilution was added to eachwell incubating for 30 min at 4 C. Cells were washed 2× in BD FACS stainbuffer BSA (BD). A staining cocktail was made by diluting the mAbs forCD16, CD4, CD19, CD45, CD3, CD56, CD14 and 1:100 in BD FACS Stainbuffer. Cells were stained with 50 ul of staining cocktail and incubatedfor 30 min at 4 C protected from light. Cells were washed 2× using BDFACS Stain buffer fixed in 100 ul of BD Stabilizing fixative. Allsamples were run with the same voltage on the BD Symphony A3 Lite. Flowcytometry data was analyzed using FlowJo 10.7.2.

TABLE 4 Flow Reagents Clone Fluorophore Supplier catalog # lot #Dilution near-IR fluorescent — near-IR Thermo L34976A 2298176  1:1,000reactive dye Fisher Human TruStain — — BioLegend 422302 B328706 1:100FcX CD16 3G8 BV 421 BioLegend 302038 B303711 1:100 CD4 OKT4 BV 605BioLegend 317438 B310529 1:100 CD19 HIB19 BV 650 BioLegend 302238B300887 1:100 CD45 HI30 BV785 BioLegend 304048 B284678 1:100 CD3 HIT3aAlexa BioLegend 300320 B278330 1:100 Fluor 488 CD56 5.1H11 PE BioLegend362508 B316093 1:100 CD14 M5E2 PE/Cy7 BioLegend 301814 B272337 1:100 CD8SK1 APC BioLegend 344722 B304311 1:100Incucyte Assay Setup

CAR-iNK and PB-NK effector cell clones were added to wells of a 96-wellflat-bottom plate (BD catalog #353072) in 100 μL of NKCM media withfinal effector number of 4×10⁵/well for 20:1 E:T wells, 2×10⁵/well for10:1 E:T wells and 2×10⁴/well for the 1:1 E:T well. NKCM assay media ismade up of 500 mL of IMDM and 500 mL Ham's F-12 Nutrient Mix as basemedia. Base media is supplemented with 2%, CTS B-27 Supplement,XenoFree, w/o Vitamin A, 1% MEM Non-Essential Amino Acids Solution, 250μM Ascorbic acid Mg 2-phosphate, 100 μM Mono-Thio-Glycerol, 1% GlutaMaxand 2 mM Nicotinamide.

Subsequently 2×10⁴K562-NLR cells was added to each well in 100 μL ofNKCM media. Assay was conducted in triplicate wells for each CAR-iNKeffector cell. Assay plates were rested at room temperature for 15minutes to allow cells to settle. Plates were placed in Incucyte S3Instrument in a 37° C. incubator with 5% CO2. Instrument scan type wasset to whole well read, with image phase, and red channel with a redacquisition time of 400 ms. Instrument scan frequency was set to readevery 3 hours for 72 hours. Whole Well Analysis parameter was selectedfor the Incucyte assay. RCU threshold was set to 2.0, and radius was setto 100 μm, with Edge Split On.

Analysis

NLR Count Per Well data was exported from Incucyte 2020A software forall wells at all timepoints, and pasted to Microsoft Excel. Eachtriplicate value was divided by the average (N=3) of the appropriateTarget Cell Only wells for each target cell line, and this value wasmultiplied by 100 to calculate values for Normalized Target Count as apercentage of the average of NLR count in target cell only wells. Datawere graphically represented with GraphPad Prism software (Version 8.)Triplicate Normalized NLR Target Count values were graphed on Y axis foreach timepoint on X axis.

Results

iNK1248-iPSC611 and PB-NK cells showed ability to kill K562 cells ateffector to target ratios of 20:1, 10:1 and 1:1. As shown in FIGS. 7A-C,the Incucyte based assay measured the loss of Nuclight Red K562 cellsover time with effector to target ratios of (A) 20:1, (B) 10:1, and (C)1:1. Normalized target cell count as a percentage of target cell onlycount for four iNK1248-iPSC611 and the average of 3 PB-NKs.

With respect to purity, the PB-NK cells were between 87% and 96%CD45+/CD56+ post isolation. iNK1248-iPSC611 was 98.8% CD45+/CD56+. PB-NKcells were between 20.15% and 0.195% CD3+ and between 19.1% and 0.075%CD3+/CD56+. iNK1248-iPSC611 was 0.08% Cd3+ and 0.048% CD3+ CD56+ (FIG. 8).

Example 9. In Vitro Elimination of CD19+ Cells Using CAR-iNK Cells

CAR-iNK clone, iNK1248-iPSC611, has have been engineered to expressanti-CD19 chimeric antigen receptor (CAR) to target CD19+ cancers. TheIncucyte live cell imaging platform was used to demonstrate thecytolytic activity of iNK1248-iPSC611 at multiple effector to targetratios (E:T) through real-time quantification of fluorescently labeledtarget cells, depletion of which serves as a measurement of efficacy ofeffector target cell killing. Isogenic pairs of Reh and NALM6 celllines, naively expressing CD19 or CD19 knock out (KO), were used todemonstrate CAR mediated lysis of CD19+ target cells.

NucLight Red Transduction of Target Cell Lines

Reh and NALM6 cells were obtained from ATCC. Cell lines were transducedwith Incucyte NucLightRed Lentivirus Reagent (EF1α Promoter, PuromycinSelection) according to manufacturer's protocol at MOI of 3, in cellculture media with 8 μg/mL Polybrene. NLR-transduced cells were selectedand cell lines were cultured in RPMI with 10% FBS and 1 μg/mL Puromycin.

Generation of Reh-CD19KO and NALM6-CD19KO Target Lines

Reh-CD19KO and NALM6-CD19KO were generated using the Lonza CRISPR-Cas-9system on parental Reh and NALM6 cells (previously NucLight Redtransduced) according to manufacturer's protocol for Amaxa 4-DNucleofector. Target sequence for custom CD19KO crRNA used was:GCTGTGCTGCAGTGCCTCAA. CD19+ cells were removed using Human CD19 PositiveSelection Kit II according to manufacturer's protocol, and CD19expression was assayed via flow cytometry. Cell lines were cultured inRPMI-10% FBS, 1 μg/mL puromycin.

TABLE 5 CD19KO Reagents Material Supplier Part# Puromycin GibcoA11138-03 Dihydrochloride SF Cell Line 4D- Lonza V4XC-2012 NucleofectorX Kit L Alt-R CRISPR- IDT 1072532 Cas9 tracrRNA Alt-R S.p. HiFi IDT1081061 Cas9 Nuclease V3 Alt-R CRISPR- IDT Custom order# Cas9 CrRNA17424545 EasySep Human Stem Cell 17854 CD19 Positive TechnologiesSelection Kit IIIncucyte CAR-iNK Killing Assay Setup

2×10⁵ (10:1), 1×10⁵ (5:1), 2×10⁴ (1:1), or 4×10³ (1:5) of each CAR-iNKeffector cell clone were added to wells of a 96-well flat-bottom plate(BD catalog #353072) in 100 μL of NKCM media, followed by 2×10⁴ Reh-NLR,Reh-CD19KO-NLR, NALM6-NLR, or NALM6-CD19KO-NLR cells in 100 μL of NKCMmedia. Assay was conducted in triplicate wells for each CAR-iNK effectorcell. Assay plates were rested at room temperature for 15 minutes toallow cells to settle. Plates were placed in Incucyte S3 Instrument in a37° C. incubator with 5% CO2. Instrument scan type was set to whole wellread, with image phase, and red channel with a red acquisition time of400 ms. Instrument scan frequency was set to read every 2 hours for 72hours. Whole Well Analysis parameter was selected for the Incucyteassay. RCU threshold was set to 2.0, and radius was set to 100 with EdgeSplit On.

Analysis

NLR Count Per Well data was exported from Incucyte 2020A software forall wells at all timepoints, and pasted to Microsoft Excel. Eachtriplicate value was divided by the average (N=3) of the appropriateTarget Cell Only wells for each target cell line, and this value wasmultiplied by 100 to calculate values for Normalized Target Count as apercentage of the average of NLR count in target cell only wells. Datawere graphically represented with GraphPad Prism software (Version 8.)Triplicate Normalized NLR Target Count values were graphed on Y axis foreach timepoint on X axis.

Results

Antigen specific lysis of both Reh and NALM6 cells by iNK1248-iPSC611cells was exhibited across a range of effector to target ratios. At eachE:T ratios tested CD19+ cells were killed quicker and more completelythan the matched CD19KO lines.

Four E:T ratios showed a range of cytolytic activity against Reh cells(FIG. 9 ). Less cytolytic activity observed in Reh CD19KO cells ascompared to parental Reh cells. FIG. 9 shows the results of an Incucytebased assay measuring the loss of Nuclight Red target cells over timewith four effector to target ratios. Normalized target cell count in Rehand Reh-CD19KO co-cultured with iNK1248-iPSC611 at E:T ratios of (A)10:1, (B) 5:1, (C) 1:1, and (D) 1:5 as a percentage of target cell onlycounts. FIG. 10 shows the results of an Incucyte based assay measuringthe loss of Nuclight Red target cells over time with four effector totarget ratios. Normalized target cell count in NALM6 and NALM6-CD19KOco-cultured with iNK1248-iPSC611 at E:T ratios of (A) 10:1, (B) 5:1, (C)1:1, (D) and 1:5 as a percentage of target cell only counts.

Example 10. In Vitro Persistence of iNK Cells

The single cell iNK clone iNK1248-iPSC611 was engineered to secrete theNK homeostatic cytokine IL-15. In a 21-day persistence assay, in thepresence of varying levels of IL-2 (10 nM-0 nM), the in-vitropersistence of iNK1248-iPSC611 was compared to that of a bulk nonengineered “wild type” (WT) iNK iNK1487-iPSC005 cell and the NK cellleukemia line KHYG-1. Every 3-4 days cells were harvested counted on aViCell Blue and re-seeded in fresh media. Cumulative fold expansion wascalculated using viable cell counts collected from the ViCell Blu.

21-Day Persistence Assay

1.5×10⁶ of iNK1248-iPSC611, WT iNK1487-iPSC005 or KHYG-1 immortalized NKcells were added to individual wells of a 24 well plate at 0.5e6/mL in atotal of 3 mL of NKCM containing six different concentrations of IL2.1.5×10⁶KHYG-1 immortalized NK cells were also added to individual wellsof a 24 well plate at 0.5e6/mL in a total of 3 mL of RPMI+10% HIFBS+1×Pen Strep containing six different concentrations of IL2. Bothplates were transferred to an incubator set at 37° C. with 5% CO2.

Every 72 or 96 hours, cells were harvested and transferred intoindividual 15 mL conical tubes. Cells were centrifuged at 300 g for 10minutes. Supernatants were aspirated, and cell pellets were resuspendedin 3 mL of basal RPMI assay media. Two hundred microliters of cells wereremoved to count on the ViCell Blu.

After counting, the cells were centrifuged again at 300 g for 10minutes. Supernatants were aspirated and cells were resuspended in NKCMassay media or RPMI assay media containing the correspondingconcentration of IL2 at 0.5e6 cells/mL. Cells were replated at 0.5e6cells/mL in 3 mL per well. If cells were resuspended at 0.5e6 cells/mLin a volume less than 3 mL, the total volume was plated. If resuspensionvolume fell below 200 uL, the cell line was not re-plated. At the end of14 days, the assay was terminated and the cells were discarded.

Analysis

Cell counts and cell viabilities were taken using the ViCell Blu. Thepopulation used to calculate fold change was viable cells per mL. Datawere graphically represented with GraphPad Prism software (Version8.4.3).

Results

The single cell clone iNK1248-IPSC611 persisted in-vitro longer than theWT iNK1487-iPSC005 in the absence of exogenous IL-2 (FIG. 11 ). Cellswere cultured in basal NKCM for 14 days at 37° C. with 5% CO2. Every 3-4days, all conditions were harvested, counted on the ViCell Blu,resuspended at 0.5e6/mL in appropriate media and then replated. After 21days, cumulative fold change was calculated. The single cell cloneiNK1248-IPSC611 persisted in-vitro longer than the WT iNK1487-iPSC005 inthe absence of exogenous IL-2 indicating that the IL-15 transgene isfunctional and exhibits the intended mode of action, namely enhancedpersistence. The IL-15 released by iNK1248-IPSC611 is adequate tosupport homeostatic survival of the cells but not sufficient to causemitogenic expansion.

Exogenous IL-2 support increased the persistence of both iNK1248-iPSC611and WT iNK1487-iPSC005 (FIG. 12A-F). Cells were cultured in NKCMcontaining one of six IL2 concentrations: 10 nM (FIG. 12A), 3 nM (FIG.12B), 1 nM (FIG. 12C), 0.3 nM (FIG. 12D), 0.1 nM (FIG. 12E), 0 nM (FIG.12F) for 21 days at 37° C. with 5% CO2. Every 3-4 days, all conditionswere harvested, counted on the ViCell Blu, resuspended at 0.5e6/mL inappropriate media and then replated. After 21 days, cumulative foldchange was calculated. Exogenous IL-2 support increased the persistenceof both iNK1248-iPSC611 and WT iNK1487-iPSC005 indicating thatadditional homeostatic cytokine is required to enable limited mitogenicexpansion of iNK1248-IPSC611. To determine if a combination ofengineered M-15 and exogenous IL-2 elicit uncontrolled proliferation oftherapeutic iNK, culture of the cells for two weeks in the presence ofIL-2 was performed and iNK1248-IPSC611 was compared to theIL-2-dependent NK leukemia line KHYG-1. KHYG-1 but not iNK1248-IPSC611exhibited logarithmic growth over two weeks of culture.

Example 11. In Vitro Elimination of Therapeutic iNK Cells with Cetuximab

Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism of cellimmune defense whereby a target cell which has been coated withantibodies recognizing cell surface antigens is lysed by an effectorcell bearing Fc receptors. ADCC can be mediated by a variety of immunecells, including natural killer (NK) cells, neutrophils, macrophages,and eosinophils by recognition of bound immunoglobulin via their Fcreceptors, particularly CD16 (FcγRIII).

Cetuximab is a chimeric mouse-human antibody targeted against theextracellular domain of epidermal growth factor receptor (EGFR). It hasbeen demonstrated to mediate ADCC against EGFR-expressing tumor celllines via its human IgG1 Fc region (Kurai, 2007)

The following experiment was conducted to assess whether theiPSC-derived NK (iNK) development candidate 611 (e.g., therapeutic iNK)expresses EGFR and is susceptible to ADCC mediated by Cetuximab comparedwith an isotype control antibody when cultured with interleukin (IL)-2activated peripheral blood mononuclear cells (PBMC).

Primary Effector Cell Isolation & Culture

Peripheral mononuclear blood cells (PBMC) were collected from buffycoats of consented healthy adult donors (Bloodworks Northwest) bycentrifugation over a Ficoll-Hypaque density gradient. Cells werecultured overnight at 1×10⁶/mL in RPMI (Life Technologies) supplementedwith 10% fetal bovine serum (FBS, Hyclone) and 55 mM b-mercaptoethanol(Life Technologies) in the presence of 10 ng/mL IL-2 (Peprotech) beforeuse in experiments.

ADCC Assays

iNK cells were labeled with 2.5 mM CTV (Life Technologies) and 2.5×10⁴cells plated/well as targets in a 96 well flat bottom plate (Corning) intriplicate. Cetuximab (Selleckchem) or a human IgG1 isotype control(Invivogen) were pre-incubated with therapeutic iNK targets atconcentrations of 10 pg/mL-10 mg/mL for 30′ prior to addition ofeffector cells. IL-2 activated effector PBMCs were added at aneffector:target (E:T) ratio of 25:1 in triplicate wells/condition andcultures incubated for 16 hours in a 5% CO2, 37% C° incubator. Deadcells were identified by flow cytometry using LIVE/DEAD™ Fixable Near-IRDead Cell Stain (ThermoFisher) according to manufacturer's protocol.Samples were acquired on a Symphony A3 (BD Biosciences) and analyzed onFlowJo version 10.7.1 software.

Flow Cytometry

For determination of antibodies bound per cell (ABC), 2×10⁵ therapeuticiNK cells were labeled with EGFR-PE (Novus Biologicals) for 15′ at RT inthe dark, washed with Cell Staining Buffer (BioLegend), and fixed for10′ at RT in the dark with Fixation Buffer (BioLegend). A single tube ofBD Quantibrite beads (BD Biosciences) was reconstituted with 500 mL PBSper manufacturer's protocol. Labeled therapeutic iNK cells and a BDQuantibrite PE tube were acquired on a Symphony A3 (BD Biosciences)using the same voltages and settings, and all samples were analyzed onFlowJo version 10.7.1 software. By using known ratios of PE toantibodies, PE molecules can be converted per cell to antibodies percell. Quantibrite beads were gated on by FSC-A vs SSC-A. Subsequentlythe PE fluorescence was visualized as a histogram and gates were drawnfor each of the 4 distinct peaks. Geometric mean fluorescence wasexported for each PE peak and used for ABC calculations.

For analysis of ADCC assays, cells were transferred to a 96 well roundbottom plate (Falcon), washed in 1×PBS pH 7.2 (Life Technologies) andresuspended in PBS containing LIVE/DEAD™ Fixable Near-IR Dead Cell Stain(ThermoFisher) according to manufacturer's protocol. Non-specificbinding to Fc receptors (FcR) was blocked using Human TruStain FcX Fcreceptor blocking solution (BioLegend) prior to addition of antibodies.Cells were incubated with antibodies against CD56 and CD16 for 20′ at RTand washed three times with Cell Staining Buffer (BioLegend) beforefixation with Fixation Buffer (BioLegend). Samples were collected on aSymphony A3 (BD Biosciences) and all FCS files analyzed on FlowJoversion 10.7.1 software.

Lymphocytes were gated on based on forward scatter area (FSC-A) and sidescatter area (SSC-A). Singlets were excluded based on forward scatterarea (FSC-A) vs forward scatter height (FSC-H) gate. Gates were drawn onCTV⁺ therapeutic iNK targets or CTV⁻ effector cells, and a subsequentgate drawn on CTV⁺ therapeutic iNK cells that labeled positive forLIVE/DEAD Fixable Near-IR. As shown in FIG. 13 , cells were gated onlymphocytes, followed by exclusion of doublets, followed by gating onCellTrace Violet (CTV)+ iNK, and finally on LIVE/DEAD™ Near-IR+ todetermine % of dead therapeutic iNK targets. FSC-A=forward scatter area,SSC-A=side scatter area, FSC-H=forward scatter height, CTV=CellTraceViolet, NIR=Near-IR.

Analysis

To calculate antibodies bound per cell (ABC), a linear regression wasplotted of Log10 PE molecules per bead against Log10 geometric mean-PE,using the following equation: y=mx+c where y equals Log10 fluorescenceand x equals Log10 PE molecules per bead. For each sample the number ofantibodies bound per cells was determined by using the equation aboveand interpolating the ABC value based on the geometric mean fluorescencevalue for each sample.

Percent specific cell lysis for ADCC assays was calculated as in (Kim,2007) using the following equation where LIVE/DEAD NIR⁺CTV⁺ targets areconsidered dead iNK and the percent of spontaneous iNK cell death isdetermined by iNK cells cultured without addition of effector cells (0:1E:T):

Results

ABC value for therapeutic iNK was calculated to be 7,341 by Quantibritebead technology using geometric mean fluorescent intensity values. (FIG.14 and Table 6). FIG. 14 shows EGFR PE levels on therapeutic iNK stainedwith EGFR (black histogram) compared with unstained therapeutic iNK(gray histogram) or an unedited WT iNK (dashed line). EGFR expressionwas observed on therapeutic iNK cells by flow cytometry with values of7,341 ABC. This level of EGFR was sufficient to observe ADCC activitymediated by Cetuximab with an EC50 of 2.0 ng/mL in co-cultures of iNKwith IL-2 activated PBMC.

TABLE 6 EGFR antibodies bound per cell Geometric Geometric mean- iNKMean background ABC Therapeutic 63.1 0 0 iNK unstained (background)Therapeutic 11,214 11,150 7,341 iNK EGFR

Addition of Cetuximab to co-cultures of IL-2 activated PBMC and iNKcells mediated ADCC of therapeutic iNK targets in aconcentration-dependent fashion, with an EC₅₀ of 2.0 ng/mL (FIG. 15 ).FIG. 15 shows the percent specific cell lysis of therapeutic iNK cellsmediated by Cetuximab (black triangles) compared with human IgG1 isotypecontrol (open triangles). IL-2 activated PBMC were co-cultured withtherapeutic iNK at a 25:1 E:T ratio for 16 hours and percent specificcell death of iNK determined. Each data point is a mean of triplicatewells, error bars±standard deviation. Addition of human IgG1 isotypecontrol did not mediate ADCC of therapeutic iNK targets, although somebackground killing was observed at the highest concentration ofantibody.

Example 12. Antibody and Complement Evasion Using B2M Knockout

Allogeneic cell therapy products derived from induced pluripotent stemcells (iPSC) have the potential to be used as an off-the-shelf treatmentfor many diseases but may generate a vigorous immune response by thehost due to incompatibilities in human leukocyte antigen (HLA) genes. Inaddition to an immune response mediated by CD8 T cells to HLA Class Imolecules, some patients may have pre-existing antibodies (Ab) to thesepolymorphic proteins (1, 2). If Abs to HLA Class I molecules do exist,there is the potential for complement-mediated cytotoxicity (CDC) of theeffector cells. A strategy to eliminate binding by Abs to HLA Class Imolecules is by deletion of beta-2 microglobulin (b2M), which encodes asubunit common to HLA Class I protein and is required for cell surfaceexpression.

The CDC assay is a simple method to measure how well an Ab induces thekilling of cells in the presence of complement proteins (3). Plasma, aswell as serum contains the full spectrum of complement proteins, whichis referred to as the complement cascade. However, these molecules arelabile and as such, collected serum samples must be quickly frozenbefore use in CDC assays. As an alternative, rabbit complement can beused as a reagent in assays to substitute for human complement. Using acommon pan-HLA-ABC Ab to model a potential HLA Class I titer from apatient (4), iNK cells were tested to demonstrate the sensitivity ofwild-type (WT) HLA Class I expressing iNK cells and protection of B2Mknock out (KO) Clone 611 iNK cells from CDC.

Complement-Mediated Cytotoxicity Assay

iNK cells, WT 005 and Clone 611, were diluted to 4×10e6/mL in RPMI-1640basal media. iNK cells were seeded at 200K cells/well in apolypropylene, U-well, 96-well plate (50 uL/well). Samples were seededin triplicate. Abs were diluted in RPMI-1640 at 40 ug/mL and dispensedat 50 uL/well (10 ug/mL final). Baby rabbit complement (BRC) was thawedjust prior to use, then diluted 1:5 in RPMI-1640 and dispensed at 100uL/well (10% BRC final). Final volumes for each well was 200 uL. Forboth iNK cell types there were 4 conditions: A, No add (RPMI-1640alone); B, Isotype Ab+BRC; C, anti-HLA-ABC Ab+BRC; and D, anti-CD52Ab+BRC. Cells were then incubated for 1 hr at 37 C, 5% CO2.

After the incubation period, the plate was centrifuged at 1200 RPM for 1minute and decanted to remove the RPMI-1640 with BRC and replaced with200 uL/well RPMI-1640+10% heat-inactivated FBS. Cells were then countedusing Trypan Blue to score both live and dead cells.

Analysis

Cellular viability was graphically represented and statisticallyanalyzed using GraphPad Prism software. Statistical significance fordifferences in viability was evaluated using Student's T test.Differences between samples were considered significant when theprobability value (p) was ≤0.05.

Results

Upon thaw and centrifugation, cells were resuspended in 1 mL Easysepbuffer and counted for viability using Trypan Blue. Cells were found tohave high viability before use in the CDC assay. WT 005: 24.6×10e6/mL,95% viability. Clone 611 iNK cells: 22.6×10e6/mL, 93% viability.

Elimination of B2M from iNK cells protects from complement-mediatedcytotoxicity in the presence of Abs to HLA-ABC molecules and complement.As shown in FIG. 16 , both freshly thawed WT 005 and Clone 611 iNK cellswere found to maintain high viability when cultured for 1 hr inRPMI-1640 alone or with the isotype Ab plus BRC. In contrast, only theWT 005 iNK cells were killed in the presence of the HLA-ABC Ab plus BRCwith no effect on clone 611 iNK cells. To prove Clone 611 iNK cells werestill sensitive to complement-mediated killing, we included an Ab toCD52. Addition of the anti-CD52 Ab plus BRC resulted in the killing ofboth iNK cells.

Example 13. Comparison of CTL Activation and iNK Cell Lysis Betweenβ2M-Deficient, iPSC-Derived NK Cells and β2M-Expressing, Wild-Type iNKCells

Allogeneic cell therapy products derived from induced pluripotent stemcells (iPSC) have the potential to be used as an off-the-shelf treatmentfor many diseases, but may generate a vigorous immune response by thehost due to incompatibilities in human leukocyte antigen (HLA) genes(Lanza, et al. Nat Rev Immunol. 2019 December; 19(12):723-733). Notably,direct lysis of mismatched class I HLA-bearing cells occurs viaactivation of host CD8+ T cells that interact with the class I HLAmolecules (Felix, et al. Nat Rev Immunol. 2007 December; 7(12):942-53).Activation of host CD8 T cells is thwarted by deletion of beta-2microglobulin (β2M), which encodes a subunit common to all class I HLAgenes and is required for their surface expression (Krangel, et al.Cell. 1979 December; 18(4):979-91; and Zijlstra, et al. Nature. 1989Nov. 23; 342(6248):435-8).

Here iPSC-derived NK (iNK) which are genetically edited to beβ2M-deficient (KO) are cultured with CD8+ cytotoxic lymphocytes (CTL)derived from peripheral blood mononuclear cells (PBMC) from multipledonors to determine whether they induce CTL activation and lysis of iNKcells compared with wild-type iNK which express (32M.

Generation of Effector Cytotoxic Lymphocytes (CTL)

Isolated cryopreserved peripheral mononuclear blood cells (PBMC) fromconsented healthy adult donors were purchased (StemCell Technologies)and stored in liquid nitrogen until use. CTLs with specific reactivityto the parental iPSC line were generated. Briefly, T cells were isolatedfrom 5×10⁷ PBMC with Human T cell Isolation kit (StemCell Technologies)according to manufacturer's instructions and primed three times byco-culture with parental iPSC-derived iNK cells in media with IL2,followed by another round of T cell isolation, and then expanded withImmunocult anti-CD2/CD3/CD28 stimulation reagent (StemCell Technologies)in media with IL2, IL7 and IL15. Expanded cells were cryopreserved inCS-10 (StemCell Technologies) buffer at 107 cells/ml.

Allo-Evasion Cytotoxicity and CTL Activation Assays

iNK cells were labeled with 5 μM CTV (Life Technologies) according tomanufacturer's instructions and 5×10⁴ cells plated/well as targets in a96-well U-bottom plate (Falcon) in duplicate. Cryopreserved CTLs werethawed and added at an effector:target (E:T) ratio of 5:1 in triplicatewells/condition and cultures incubated for 48 hours in a 5% CO2, 37% Coincubator.

Flow Cytometry

Cells were washed in 1×PBS pH 7.2 (Life Technologies) and resuspended inPBS containing LIVE/DEAD™ Fixable Near-IR Dead Cell Stain (ThermoFisher)according to manufacturer's protocol. Non-specific binding to Fcreceptors (FcR) was blocked using Human TruStain FcX Fc receptorblocking solution (BioLegend) prior to addition of antibodies. Cellswere incubated with antibodies against TCRab, CD4, CD8 and CD25 for 20′at RT and washed three times with Cell Staining Buffer (BioLegend)before fixation with Fixation Buffer (BioLegend). Samples were collectedon a Symphony A3 (BD Biosciences) and all FCS files analyzed on FlowJoversion 10.7.1 software.

Lymphocytes and quantitation beads were gated based on forward scatterheight (FSC-H) and side scatter area (SSC-A). Singlets were excludedbased on forward scatter area (FSC-A) vs forward scatter height (FSC-H)gate. Live cells were gated as negative for LIVE/DEAD NIR staining.Based on CTV and TCRαβ, T cells (TCRαβ positive, CTV negative) and iNKcells (CTV positive and TCRαβ negative) were gated. Within the T cellgate, CD8 positive and CD4 negative cells were selected. Within the CD8+T cell population, expression of CD25 was assessed, the CD25-positivegate determined to capture minimal positive background events among Tcells cultured alone without targets (FIG. 17 ). As shown in FIG. 17 ,cells were gated on quantitation beads and lymphocytes. Withinlymphocytes exclusion of doublets, followed by gating on LIVE/DEAD™Near-IR negative, followed by CTV to identify iNK cells and TCRαβ toidentify T cells. Within T cells, CD4-negative, CD8-positive cells,followed by CD25 to identify activated CD8+ T cells. Key assayparameters Quantitation beads, live iNK cells and activated CD8+ T cellsare indicated. FSC-A=forward scatter area, SSC-A=side scatter area,FSC-H=forward scatter height, FSC-W=forward scatter width,L-D=LIVE/DEAD™ Near-IR, CTV=CellTrace Violet.

Analysis

The live iNK number for each well was normalized by dividing theacquired CTV+ gate event count by the event count from the quantitationbead gate. Average of duplicate wells for each donor condition was usedfor calculated values. Specific lysis of iNK cells by CTL was determinedby the following calculation:

Where iNK_(a) is the normalized CTV+ event count in the given iNK:CTLco-culture condition; and iNK_(a) is the normalized CTV+ event count inthe corresponding control iNK alone condition. To determine significanceof assay outcomes, p-values were determined by unpaired student's t-testof assay values, n=3 individual donors.

Results

Specific killing of parental iNK cells was observed at 86-98%,corresponding to 64-84% activation of CTL when co-cultured with theparental iNK cells. 0.5-21% specific killing was observed among editedβ2MKO iNK, corresponding to 1-3% activation of CTL in co-culture withβ2MKO iNK cells. Both iNK killing and CTL activation were significantlyreduced when β2MKO iNK cells were used as targets.

iNK cells were incubated alone or with CTL at a 5:1 CTL:iNK ratio for 48hours, then live iNK cells were measured by flow cytometry (FIG. 18A).Parental iNK exhibited 86-98% specific lysis, while β2MKO iNK exhibited0.5-21% specific lysis (FIG. 18B).

CTL were incubated alone or with iNK at a 5:1 CTL:iNK ratio for 48hours, then activation of CD8+ T cells by CD25 expression was measuredby flow cytometry (FIG. 19A). 64-84% of CTL were activated by theparental wild-type iNK, while 1-3% were activated by β2MKO iNK, and0.5-5% activated without target cells present (FIG. 19B).

Example 14. PBMC-Mediated Killing of β2M^(−/−)/HLA-E⁺ iNK Cells

Allogeneic cell therapy products derived from induced pluripotent stemcells (iPSC) have the potential to be used as an off-the-shelf treatmentfor many diseases, but may generate a vigorous immune response by thehost due to incompatibilities in human leukocyte antigen (HLA) genes. Astrategy to eliminate activation of host CD8 T cells is by deletion ofbeta-2 microglobulin (β2M), which encodes a subunit common to class Imajor histocompatibility complex (MHC) and is required for surfaceexpression of MHC class I (Krangel, et al. Cell. 1979 December;18(4):979-91; and Zijlstra, et al. Nature. 1989 Nov. 23;342(6248):435-8).

A limitation of this approach, however, is that while rejection ofengineered iPSC cell products by CD8 may be abrogated, these MHC classI-negative cells may be lysed by natural killer (NK) cells due to“missing self” (Bix, et al. Nature 349, 329-331 (1991); Liao, et al.Science 253, 199-202 (1991)).

An approach to limit lysis by host NK cells is by overexpression ofHLA-E on the surface of iPSC-derived cell products (Gornalusse, et al.Nat Biotechnol. 2017 August; 35(8):765-772; Hoerster, et al. FrontImmunol. 2021 Jan. 29; 11:586168). HLA-E is a minimally polymorphicligand which presents peptides derived from signal sequences of otherHLA class I molecules and binds the inhibitory NK receptor complexCD94/NKG2A (Braud, et al. Nature 349, 329-331 (1991); Miller, et al. JImmunol. 2003 Aug. 1; 171(3):1369-75).

Here iPSC-derived NK (iNK) which are edited to be β2M^(−/−) but expressHLA-E (e.g., therapeutic iNK) are cultured with peripheral bloodmononuclear cells (PBMC) to determine whether they are less susceptibleto killing by PBMC compared with iNK which lack PM and do not expressHLA-E.

Primary Effector Cell Isolation and Culture

Peripheral mononuclear blood cells (PBMC) were collected from buffycoats of consented healthy adult donors (Bloodworks Northwest) bycentrifugation over a Ficoll-Hypaque density gradient and cryopreservedin Cryostor CS10.

Allo-Evasion Cytoxicity Assays

iNK cells were labeled with 2.5 μM CTV (Life Technologies) according tomanufacturer's instructions and 2.5×10⁴ cells plated/well as targets ina 96 well flat bottom plate (Corning) in triplicate. Cryopreserved PBMCswere thawed and added at an effector:target (E:T) ratio of 25:1 intriplicate wells/condition and cultures incubated for 72 hours in a 5%CO2, 37% Co incubator. Dead cells were identified by flow cytometryusing LIVE/DEAD™ Fixable Near-IR Dead Cell Stain (ThermoFisher)according to manufacturer's protocol. Samples were acquired on aSymphony A3 (BD Biosciences) and analyzed on FlowJo version 10.7.1software.

Flow Cytometry

For determination of antibodies bound per cell (ABC), 1×10⁵ iNK cellswere labeled with mouse IgG1 PE isotype control (BioLegend) or HLA-E PE(BioLegend) for 15′ at RT in the dark, washed with Cell Staining Buffer(BioLegend), and fixed for 10′ at RT in the dark with Fixation Buffer(BioLegend). A single tube of BD Quantibrite beads (BD Biosciences) wasreconstituted with 500 mL PBS per manufacturer's protocol. Labeled iNKcells and a BD Quantibrite PE tube were acquired on a Symphony A3 (BDBiosciences) using the same voltages and settings, and all samples wereanalyzed on FlowJo version 10.7.1 software. By using known ratios of PEto antibodies, PE molecules can be converted per cell to antibodies percell. Quantibrite beads were gated on by FSC-A vs SSC-A. Subsequentlythe PE fluorescence was visualized as a histogram and gates were drawnfor each of the 4 distinct peaks. Geometric mean fluorescence wasexported for each PE peak and used for ABC calculations.

For NK cell phenotyping, cells were transferred to a 96 well roundbottom plate (Falcon), washed in 1×PBS pH 7.2 (Life Technologies) andresuspended in PBS containing LIVE/DEAD′ Fixable Near-IR Dead Cell Stain(ThermoFisher) according to manufacturer's protocol. Non-specificbinding to Fc receptors (FcR) was blocked using Human TruStain FcX Fcreceptor blocking solution (BioLegend) prior to addition of antibodies.Cells were incubated with antibodies against CD3, CD56, and CD16 for 20′at RT and washed three times with Cell Staining Buffer (BioLegend)before fixation with Fixation Buffer (BioLegend). Samples were collectedon a Symphony A3 (BD Biosciences) and all FCS files analyzed on FlowJoversion 10.7.1 software.

For analysis of allo-evasion cytotoxicity assays, cells were transferredto a 96 well round bottom plate (Falcon), washed in 1×PBS pH 7.2 (LifeTechnologies) and resuspended in PBS containing LIVE/DEAD™ FixableNear-IR Dead Cell Stain (ThermoFisher) according to manufacturer'sprotocol. Non-specific binding to Fc receptors (FcR) was blocked usingHuman TruStain FcX Fc receptor blocking solution (BioLegend) prior toaddition of antibodies. Cells were incubated with antibodies againstCD56 and CD16 for 20′ at RT and washed three times with Cell StainingBuffer (BioLegend) before fixation with Fixation Buffer (BioLegend).Samples were collected on a Symphony A3 (BD Biosciences) and all FCSfiles analyzed on FlowJo version 10.7.1 software.

Lymphocytes were gated on based on forward scatter area (FSC-A) and sidescatter area (SSC-A). Singlets were excluded based on forward scatterarea (FSC-A) vs forward scatter height (FSC-H) gate. Gates were drawn onCTV⁺ iNK targets or CTV⁻ effector cells, and a subsequent gate drawn onCTV⁺ iNK cells that labeled positive for LIVE/DEAD Fixable Near-IR (FIG.20 ). Cells were gated on lymphocytes, followed by exclusion ofdoublets, followed by gating on CellTrace Violet (CTV)+ iNK, and finallyon LIVE/DEAD™ Near-IR+ to determine % of dead iNK targets. FSC-A=forwardscatter area, SSC-A=side scatter area, FSC-H=forward scatter height,CTV=CellTrace Violet, NIR=Near-IR.

Analysis

To calculate antibodies bound per cell (ABC), a linear regression wasplotted of Log10 PE molecules per bead against Log10 geometric mean-PE,using the following equation: y=mx+c where y equals Log10 fluorescenceand x equals Log10 PE molecules per bead. For each sample the number ofantibodies bound per cells was determined by using the equation aboveand interpolating the ABC value based on the geometric mean fluorescencevalue for each sample after subtraction of isotype control backgroundvalues.

Cell death for allo-evasion assays was calculated by determining themean percent of LIVE/DEAD NIR⁺CTV⁺ targets (dead iNK) for each iNK groupand dividing by the mean percent of LIVE/DEAD NIR+CTV+WT iNK targets.Results are presented as “Cell death relative to WT iNK”.

Results

HLA-E expression was measured on edited iNK cells from line 004 by flowcytometry with a value of 3.625 ABC. Expression of HLA-E on therapeuticiNK cells was sufficient to observe a reduction in cell death whencultured with PBMC compared with HLA-E negative, β2M KO iNK.

ABC value for HLA-E expressing therapeutic iNK cells was calculated tobe 3,625 by Quantibrite using geometric mean fluorescent intensityvalues. (FIG. 21 ; HLA-E=open histogram, mouse IgG1 isotype control=grayfilled histogram).

HLA-E binds the heterodimer CD94/NKG2A, an inhibitory receptor which isexpressed on NK cells. Because CD94 can also pair with NKG2C to form anactivating receptor, it was not assessed here. Frequency ofNKG2A-expressing NK cells within a PBMC milieu was measured on twodonors. Cryopreserved PBMC were thawed and stained for cells expressingNK cell markers (CD3⁻CD56⁻CD16^(+/−)) and frequencies ofNKG2A-expressing NK cells assessed. In donor 1, 63.7% of NK cellsexpressed NKG2A while donor 2 contained 44.1% NKG2A⁺ NK cells. (FIG. 22). PBMC samples were gated on viable lymphocytes, followed by a gate onCD3-CD56+ cells (“NK cells”). Frequencies of NKG2A-expressing NK cellswere then determined based on an FMO.

Donor mis-matched PBMC and edited iNK cells were incubated at a 25:1 E:Tratio for 72 hours, and iNK cell viability measured by flow cytometry.iNK cells lacking surface HLA (b2M KO, white bars) exhibited anapproximate 2.25 and 1.5-fold increase in cell death relative to WT(black bars) in PBMC co-cultures with donors 1 and 2, respectively.Therapeutic iNK cells, which express HLA-E, reduced cell death to thelevel of WT iNK (gray bars) (FIG. 23 and Table 7). Freshly thawed PBMCwere co-cultured with therapeutic iNK at a 25:1 E:T ratio in thepresence of 10 ng/mL IL-15 for 72 hours and cell death of edited iNKrelative to WT determined as described in methods. Each data point is amean of triplicate wells.

TABLE 7 Percent cell death in PBMC: Therapeutic iNK co-culturesTherapeutic WT iNK b2M KO iNK iNK Donor 1 19.63 ± 0.91 44.47 ± 2.1 18.8± 4.6 Donor 2 25.67 ± 0.67 39.67 ± 2.1 24.0 ± 0.95 Mean cell death ±standard deviation

Example 15. In Vivo Evaluation of Anti-Tumor Efficacy of iNK Cells

The purpose of this study is to evaluate the in vivo anti-tumor efficacyof cryopreserved iPSC611 CD19iNK cells. A secondary purpose of thisstudy is to evaluate single-dose 7-day persistence of cryopreservediPSC611 CD19iNK.

Animals

For this study, female NSG (NOD·Cg-Prkdc^(scid) Il2rg^(tmlWjl)/SzJ) mice(Jackson Labs, Bar Harbor, Me., USA), were used. At study initiation,mice were 7-9 weeks of age, and initial body weight was an average of 23grams. Animals were acclimated for one week prior to any experimentalprocedures being performed.

Autoclaved water and irradiated food (Laboratory Autoclavable RodentDiet 5010, Lab Diet) were provided ad libitum, and the animals weremaintained on a 12-hour light and dark cycle. Cages, bedding, and waterbottles were autoclaved before use and changed biweekly. The experimentwas carried out in accordance with The Guide for the Care and Use ofLaboratory Animals.

Tumors

NALM6-Fluc-Puro (ALL) tumor cells (Imanis Life Sciences, CL151) weremaintained in RPMI 1640 medium with 10 mM HEPES, 2.5 μg/mL Puromycin,and 10% (v/v) HI FBS. Each mouse received 1×10⁵NALM6-Fluc-Puro cells inserum-free RPMI 1640 medium in a total volume of 0.2 mL.

Efficacy Study Design & Treatment

The tumor cell implant day was designated as Study Day 0.NALM6-Fluc-Puro tumor cells were intravenously implanted, and micerandomized into treatment groups of N=10 by bioluminescent signal (range64,120-141,400 p/s/cm²/sr; mean=92,649±19,925 p/s/cm²/sr).

On Days 1, 8, and 15 following i.v. NALM6-Fluc-Puro tumor cellimplantation, mice were intravenously injected with 10×10⁶ or 15×10⁶cryopreserved iPSC611 therapeutic iNK cells thawed and resuspended inLactated Ringer's/5% Human Serum Albumin (Groups 2, 3), in a volume of0.2 mL. Group 1 remained as an untreated control (Table 8, EfficacyStudy Design).

TABLE 8 Efficacy Study Design Dose Level Group N Treatment (cells/mouse)Preparation 1 10 N/A 0 N/A 2 10 iPSC611 10 × 10⁶ Cryogenic 3 10 15 × 10⁶

All mice received intraperitoneal recombinant human IL-2 (PeproTech®200-02) on Days 1, 2, 4, 7, 8, 10, 12, 15, 17, 19, 21, 23, 25, and 28,at a dose of 100,000 international units (IU) per mouse in 0.2 mL.Briefly, lyophilized rhIL-2 (1 mg) is centrifuged at 2000 g for 1minute, resuspended and solubilized in 1 mL 100 mM acetic acid, thenmixed with 4 mL 0.1% BSA in PBS. 1 mL aliquots are frozen at −80° C.until use, at which point the aliquot is thawed at ambient temperatureand mixed with 3 mL PBS for a final concentration of 500,000 IU/mL.

Tumor burden was assessed by bioluminescent imaging using an IVIS LuminaS5 (Perkin Elmer®). Briefly, mice were injected i.p. with 150 mg/kgD-Luciferin (VivoGlo™ Luciferin, Promega™), anesthetized via 2.5-3.5%vaporized isoflurane in oxygen, and imaged on automatic exposureventrally and dorsally 20 minutes post-luciferin injection. Totalwhole-body bioluminescence is calculated by adding the average radianceof ventral and dorsal images.

Animal body weight and bioluminescence were monitored twice weekly.Animals were monitored daily for clinical signs. Individual animals wereremoved from the study and humanely euthanized when in moribundcondition, or when an animal lost ≥20% of the original body weight forthree consecutive measurements.

In some instances, supportive nutrition and hydration was provided, toensure wellness of the mice on study. All mice were provided withHydroGel® ad libitum on the day of treatment (Days 1, 8, and 15).

Persistence Study

An additional cohort of satellite animals was designated for tissuesampling to evaluate single dose persistence of iPSC611 cells. 10 femaleNSG mice were intravenously implanted with NALM6-Fluc-Puro cells asdescribed previously, on Day 0. On Day 1, mice received a singleintravenous injection of 15×10⁶ cryogenic iPSC611 cells (Group 3). Group1 remained as an untreated control Table 9, Persistence Study Design).All animals received recombinant human IL-2, dosed as describedpreviously, on Days 1, 3, 5, and 7.

TABLE 9 Persistence Study Design Dose Level Group* N Treatment(cells/mouse) Preparation 1 5 N/A 0 N/A 3 5 iPSC611 15 × 10⁶ Cryogenic*Groups numbered to match those of efficacy study

On Day 8, all mice on study plus one naïve age-matched mouse, werehumanely euthanized and sampled. Whole blood was collected via cardiacpuncture into lithium heparin-coated tubes (BD 365965). Lungs wereflushed with PBS through the right ventricle in situ, trimmed, andplaced into 2.4 mL 1× Buffer S (Miltenyi Biotech GmbH, 130-095-927) onwet ice until processing. Cervical lymph nodes were harvested and placedinto 2.4 mL 1× Buffer S on wet ice until processing.

Blood was processed by transferring to a 96 well 2 mL deep well platecontaining 1.5 mL of PBS. The plate was centrifuged for 5 min at 300 gand supernatant was decanted. The cell pellets were resuspended in 750μL of ACK lysis solution and incubated at room temp for 5 min, at whichpoint 750 μL of PBS was added to each well. The plate was centrifugedfor centrifuged for 5 min at 300 g and supernatant was decanted. ACKlysis was repeated 2× as described above. After completion of ACK lysisthe resulting Cell pellets were resuspended in 1500 of PBS andtransferred to a 96 well U bottom plate for FACS staining and analysis.

TABLE 10 FACS Reagents Clone Fluorophore Supplier catalog # lot #Dilution LIVE/DEAD ™ Fixable Near-IR L34976A  1:1000 Near-IR viabilitydye Fc Receptor Blocker Innovex NB309 1:2  CD45 HI30 BV421 Biolegend304032 B286533 1:20 CD56 5.1H11 BV786 Biolegend 362550 B303958 1:20

Tissues were processed using the Miltenyi Biotech GmbH Lung DissociationKit. Briefly, 1× Buffer S was prepared by mixing 1 mL 20× Buffer S with19 mL sterile water. Enzyme D was reconstituted with 3 mL 1× Buffer S,using gentle inversion every minute until solubilized. Enzyme A wasreconstituted with 1 mL 1× Buffer S, using gentle inversion every minuteuntil solubilized. Tissues were individually collected into 1× Buffer Sin gentleMACS C Tubes. Immediately prior to processing, 100 μL of EnzymeD and 15 μL of Enzyme A were added to each tube. Tubes were placed onthe gentleMACS Dissociator on program “m_lung_01.” The tubes were thenplaced in incubation at 37 C on the MACSmix Tube Rotator for 30 minutes,followed by further mechanical dissociation using the gentleMACSDissociator on program “m_lung_02.” Samples were then filtered through aMACS SmartStrainer (70 μm) placed on a 50 mL tube and washed with 10 mLPBS. The suspension was centrifuged at 300×g for 10 minutes, supernatantaspirated, and cell pellet resuspended in PBS at 10×10⁶ cells/mL forplating, staining, and FACS analysis.

Cell suspensions from blood, lung and cervical lymph nodes were platedat approximately 1e6 cells per well in a 96-well U-bottom plate (BDfalcon 353077). All wash steps carried out by centrifugation at 300×Gfor 3 min and flicking supernatants into the sink. Cells were washed 2×in PBS and stained with 50 μl of a 1:1000 dilution (in PBS) ofLIVE/DEAD™ Fixable Near-IR viability dye (thermo Fisher) for 15 min atroom temperature (RT). 500 of Fc Receptor Blocker (Innovex NB309) wasadded to each well and incubated for 20 minutes at 4° C. Cells werewashed 2× in BD FACS stain buffer BSA (BD). A staining cocktail weremade by diluting the mAbs for CD45 and CD56 1:20 in BD FACS Stainbuffer. Cells were stained with 500 of staining cocktail and incubatedfor 30 min at 4 C protected from light. Cells were washed 2× using BDFACS Stain buffer fixed in 100 μl of BD Stabilizing fixative. Allsamples were run with the same voltage on the BD Symphony A3 Litecollecting all events. Flow cytometry data was analyzed using FlowJo10.7.2.

iNK cells were defined as live singlets that were CD45+ and CD56+ andrepresented as #iNK cells per 100K live lymphocytes. The lower limit ofdetection (LLOD) was defined as maximum+1 standard deviation (SD) of thecontrol group that received no iNK treatment. Samples above the LLODwere plotted in graph pad Prism.

Analysis

Body weights are graphically represented as percent change in mean groupbody weight, using the formula: where ‘W’ represents mean body weight ofthe treated group on a particular day, and ‘W₀’ represents mean bodyweight of the same treated group at initiation of treatment.

Percent tumor growth inhibition (TGI) is defined as the differencebetween whole body average radiance of the treated and control groups,calculated as % TGI=(I−T/C) 100 where T is the average radiance of thetreatment group and C is the average radiance of the control group.

For survival assessment, results are plotted as the percentage survivalagainst days post-tumor implantation. Adverse clinical signs indicatingexcessive tumor burden (such as ruffled/matted fur, hunched posture,inactivity, or hind limb weakness) are used as a surrogate endpoint fordeath. Median survival is determined utilizing Kaplan Meier survivalanalysis.

The percent increased lifespan (ILS) is calculated as % ILS=S_(T)/S_(C),where S_(T) is the median survival day of the treatment group and S_(C)is the median survival day of the control group. Animals failing toreach the surrogate endpoint due to adverse clinical signs or deathunrelated to treatment or tumor burden, are censored for the survivalassessment.

Tumor bioluminescent data, body weight, survival, and persistence weregraphically represented and statistically analyzed utilizing GraphPadPrism software (Version 9.0.1). Statistical significance for tumorbioluminescence was evaluated using an ordinary two-way analysis ofvariance (ANOVA) and Tukey multiple comparisons, with a 95% confidenceinterval. Differences between groups were considered significant whenthe probability value (p) was ≤0.05. Statistical significance forprobability of survival was evaluated using a Mantel-Cox test with aGehan-Breslow-Wilcoxon test. Statistical significance for persistencewas evaluated using an ordinary one-way analysis of variance (ANOVA) andTukey multiple comparisons, with a 95% confidence interval. Differencesbetween groups were considered significant when the probability valuewas ≤0.05.

Results

Cryogenic iPSC611 cells were well-tolerated as determined by body weightand clinical observations. iPSC611 demonstrated significant anti-tumorefficacy at both dose levels. Enhanced increased life span was observedin mice treated with iPSC611 cells. Cryogenic iPSC611 had limited invivo persistence one week post-injection.

Group mean body weight changes of NALM6-Fluc-Puro tumor-bearing micetreated with iPSC611 cells or tumor alone control, are graphicallyrepresented in FIG. 24 (Mean percent body weight change of untreatedmice (•), or mice treated intravenously with iPSC611 at 10×10⁶ (▾) and15×10⁶ (♦) (cryogenic) cells). Means are plotted where ≥50% of thetreatment group are present. Arrows represent dosing days. Nosignificant body weight loss (>10% loss from the start of treatment) wasobserved in any group receiving iPSC611 cells or in the tumor alonecontrol.

Statistically significant anti-tumor activity was observed with iPSC611at 10×10⁶ and 15×10⁶ cryogenic cells (Table 11). Tumor growth isrepresented in FIG. 25 (Mean whole body average radiance of untreatedmice (•), and mice treated intravenously weekly for three doses withiPSC611 at 10×10⁶ (▾) and 15×10⁶ (♦) (cryogenic) cells). Groups areplotted until Day 21, the last imaging timepoint where the untreatedcontrol group remained and the timepoint at which % TGI was calculated.Arrows represent dosing days.

TABLE 11 Tumor Growth Inhibition of iPSC611 in the Intravenous NALM6Xenograft Model 10 × 10⁶ cryogenic 15 × 10⁶ cryogenic cells/mousecells/mouse Treatment p-value TGI (%) p-value TGI (%) iPSC611 0.023066.7 0.0089 75.6 ^(a) Only p-values ≤ 0.05 (significant with 95%confidence interval) are reported. NS = not significant; TGI = tumorgrowth inhibition.

Percent Increased Life Span (% ILS) was calculated for all treatmentgroups. Enhanced survival over tumor alone control was observed forgroups receiving iPSC611 at 10×10⁶ and 15×10⁶ cryogenic cells (Table 12,FIG. 26 ).

TABLE 12 Percent Increased Life Span for NALM6-bearing mice treated withiPSC611 Treatment Condition Dose % ILS iPSC611 Cryogenic 10 × 10⁶ 109.115 × 10⁶ 113.6

Persistence of fresh and cryogenic iPSC611 was evaluated in blood andtissue, one week following injection of iNK into NALM6 tumor-bearingmice. Poor recovery of live cells was observed for cervical lymph nodesamples. Therefore, these were not analyzed.

FACS analysis of lungs and blood indicated limited persistence ofcryogenic iPSC611 (FIG. 27 ). Mice were left untreated, or received asingle intravenous dose of iPSC611 at 15×10⁶ cryogenic cells. One-weekpost-injection, lungs and blood were harvested for FACS analysis. Numberof iNK per 100,000 lymphocytes is plotted for individual mice (o), andaverage per group represented by bars. iNK were detected in lungs andblood of two of the five mice injected with iPSC611, one weekpost-injection.

Example 16. In Vivo Evaluation of Elimination of iNK Cells

The purpose of this study is to evaluate the in vivo elimination ofcryopreserved iPSC611 CD19iNK cells, using Erbitux (cetuximab).

Animals

For this study, female NSG (NOD·Cg-Prkdc^(scid) Il2rg^(tmlWjl)/SzJ) mice(Jackson Labs, Bar Harbor, Me., USA), were used. At study initiation,mice were 10-12 weeks of age, and initial body weight was an average of24.3 grams. Animals were acclimated for one week prior to anyexperimental procedures being performed.

Autoclaved water and irradiated food (Laboratory Autoclavable RodentDiet 5010, Lab Diet) were provided ad libitum, and the animals weremaintained on a 12-hour light and dark cycle. Cages, bedding, and waterbottles were autoclaved before use and changed biweekly. The experimentwas carried out in accordance with The Guide for the Care and Use ofLaboratory Animals.

Study Design and Treatment

Mice were randomized into groups of N=5 by body weight (range 23.1-25.7grams; mean=24.3±0.85 grams) (Table 13, Study Design).

The iPSC611 cell implant day was designated as Study Day 1. On Day 1,mice were intravenously injected with 15×10⁶ cryopreserved iPSC611 cellsthawed and resuspended in Lactated Ringer's/5% Human Serum Albumin(Groups 2, 3), in a volume of 0.2 mL. Group 1 remained as an untreatedcontrol.

All mice in Groups 1, 2, and 3 received intraperitoneal recombinanthuman IL-2 (PeproTech® 200-02) on Days 1 and 3, at a dose of 100,000international units (IU) per mouse in 0.2 mL. Briefly, lyophilizedrhIL-2 (1 mg) is centrifuged at 2000 g for 1 minute, resuspended andsolubilized in 1 mL 100 mM acetic acid, then mixed with 4 mL 0.1% BSA inPBS. 1 mL aliquots are frozen at −80° C. until use, at which point thealiquot is thawed at ambient temperature and mixed with 3 mL PBS for afinal concentration of 500,000 IU/mL.

On Days 2 and 3, mice received intraperitoneal antibody therapy. Group 2was dosed with 20 mL/kg PBS, IP. Group 3 was dosed with 40 mg/kgcetuximab in a volume of 20 mL/kg, IP.

Animal body weight was recorded daily. Animals were monitored daily forclinical signs.

TABLE 13 Study Design Day 2 and 3 Day 1 Treatment (IP) CD19iNK DoseLevel Group N (IV) Agent (mg/kg) rhIL-2 (IP) 1 2 N/A N/A N/A + 2 5iPSC611 PBS 0 + 3 5 15 × 10⁶ cetuximab 40 + cells/mouse IV = intravenousIP = intraperitoneal + = dosedSampling

On Day 5, all mice on study were humanely euthanized and sampled. Bloodwas collected via cardiac puncture into Lithium Heparin coated tubes (BDMicrotainer 365965). Lungs were flushed with PBS through the rightventricle in situ, trimmed, and placed into PBS+2% FBS on wet ice untilprocessing.

Blood was processed through 2 rounds of ACK lysis following thefollowing protocol. Blood was transferred to a 2 ml deep well plate andtubes rinsed with 1 ml of PBS. Deep well was centrifuged for 3 min at300×G. The supernatant was removed and 1 mL of ACK added to each well.The plate was incubated for 2 minutes and then 1 mL of PBS was added tostop osmotic lysis. The plate was centrifuged for 3 minutes at 300×G andsupernatant was removed. ACK lysis was repeated 1-2 more times asneeded. Samples were resuspended in 200 μL of BD FACS stain buffer andtransferred to a 96 well U-bottom plate for staining

Lungs were processed to a single-cell suspension using mechanicaldissociation and gentle enzymatic digestion. Briefly, lung tissue wastransferred into a dish without medium, and minced into a homogenouspaste (<1 mm in size) using a razor blade or scalpel. Minced tissue wastransferred to 2 mL digestion medium containing 10%Collagenase/Hyaluronidase, 15% DNase I Solution (1 mg/mL), and 75% RPMI1640 Medium, and incubated at 37° C. for 20 minutes on a shakingplatform. The tissue was then passed through a 70 μm nylon mesh strainerover a 50 mL conical tube using the rubber end of a syringe plunger toobtain a cell suspension. The suspension was passed through a new 70 μmnylon mesh strainer over a 50 mL conical tube to filter, and rinsed with10 mL RPMI. The cell suspension was transferred into a 15 mL conicaltube and centrifuge at 500×G for 10 minutes at room temperature with thebrake on low. Supernatant was removed and discarded. Cells wereresuspended in 10 mL of PBS and counted, adjusted to 10×10⁶ cells/mL,and underwent one ACK lysis step before plating, staining, and FACSanalysis.

TABLE 14 FACS Reagents Clone Fluorophore Supplier catalog # lot #Dilution LIVE/DEAD ™ Fixable Near-IR L34976A  1:1000 Near-IR viabilitydye Fc Receptor Blocker Innovex NB309 1:2  CD45 HI30 BV421 Biolegend304032 B286533 1:20 CD56 5.1H11 BV786 Biolegend 362550 B303958 1:20

Cell suspensions from lung were plated at approximately 1e6 cells perwell in a 96-well U-bottom plate (BD falcon 353077). All wash stepscarried out by centrifugation at 300×G for 3 minutes and flickingsupernatants into the sink. Cells were washed 2× in PBS and stained with50 μl of a 1:1000 dilution (in PBS) of LIVE/DEAD™ Fixable Near-IRviability dye (thermo Fisher) for 15 minutes at room temperature (RT).50 μl of Fc Receptor Blocker (Innovex NB309) was added to each well andincubated for 20 minutes at 4° C. Cells were washed 2× in BD FACS stainbuffer BSA (BD). A staining cocktail were made by diluting the mAbs forCD45 and CD56 1:20 in BD FACS Stain buffer. Cells were stained with 50μl of staining cocktail and incubated for 30 minutes at 4° C. protectedfrom light. Cells were washed 2× using BD FACS Stain buffer fixed in 100μl of BD Stabilizing fixative. All samples were run with the samevoltage on the BD Symphony A3 Lite collecting all events. Flow cytometrydata was analyzed using FlowJo 10.7.2.

iNK cells were defined as live singlets that were CD45+ and CD56+ andrepresented as #iNK cells per 100K live lymphocytes. The lower limit ofdetection (LLOD) was defined as maximum+1 standard deviation (SD) of thecontrol group that received no iNK treatment. Samples above the LLODwere plotted in graph pad Prism.

Analysis

Body weights are graphically represented as percent change in mean groupbody weight, using the formula: where ‘W’ represents mean body weight ofthe treated group on a particular day, and ‘W₀’ represents mean bodyweight of the same treated group at initiation of treatment.

Body weight and persistence were graphically represented andstatistically analyzed utilizing GraphPad Prism software (Version9.0.1). Statistical significance for elimination was evaluated using anunpaired one-tailed t-test with Welch's correction, with a 95%confidence interval. Differences between groups were consideredsignificant when the probability value was ≤0.05.

Results

iPSC611 cells were significantly reduced in the lungs and blood of micethat received cetuximab treatment. Group mean body weight changes ofmice are graphically represented in FIG. 28 (mean percent body weightchange of mice treated intravenously with iPSC611 at 15×10⁶ cellsreceiving IP PBS (•), or cetuximab at 40 mg/kg (▪)). No significant bodyweight loss (>10% loss from the start of treatment) was observed in anygroup receiving iPSC611 cells and antibody.

The presence of iNK was evaluated in blood and lungs, four daysfollowing injection of iPSC611 into NSG mice. FACS analysis of lungsindicated a significant 96% reduction in number of iNK in the lungs ofmice that received cetuximab versus PBS-treated mice (p=0.0002). Asshown in FIG. 29 , FACS analysis of blood indicated a significant 95%reduction in number of iNK in the blood of mice that received cetuximabversus PBS-treated mice (p=0.0321).

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the present description.

It is claimed:
 1. A polynucleotide encoding an inactivated cell surfacereceptor that comprises a truncated epithelial growth factor receptor(tEGFR) variant comprising an amino acid sequence having at least 90%sequence identity to SEQ ID NO: 71 and an interleukin 15 (IL-15)comprising an amino acid sequence having at least 90% sequence identityto SEQ ID NO. 72, wherein the tEGFR variant and the IL-15 are operablylinked by an autoprotease peptide comprising the amino acid sequence ofSEQ ID NO:
 73. 2. The polynucleotide according to claim 1, wherein thetEGFR variant consists of the amino acid sequence of SEQ ID NO:
 71. 3.The polynucleotide according to claim 1, wherein the IL-15 consists ofthe amino acid sequence of SEQ ID NO:
 72. 4. The polynucleotideaccording to claim 1, wherein the autoprotease peptide consists of theamino acid sequence of SEQ ID NO:
 73. 5. The polynucleotide according toclaim 1 consisting of operably linked polynucleotides encoding the tEGFRvariant having the amino acid sequence of SEQ ID NO: 71, theautoprotease peptide having the amino acid sequence of SEQ ID NO: 73,and the IL-15 having the amino acid sequence of SEQ ID NO:
 72. 6. Thepolynucleotide of claim 1, wherein the inactivated cell surface receptorcomprises the amino acid sequence of SEQ ID NO:
 74. 7. A protein encodedby the polynucleotide according to claim
 1. 8. An induced pluripotentstem cell (iPSC) or a derivative cell thereof comprising thepolynucleotide of claim
 1. 9. A vector comprising the polynucleotideaccording to claim
 1. 10. The vector according to claim 9, wherein thevector further comprises: (i) a promoter; (ii) a terminator and/or apolyadenylation signal sequence; (iii) a left homology sequencecomprising a polynucleotide sequence having at least 90% sequenceidentity to the polynucleotide sequence of SEQ ID NO: 84; and (iv) aright homology sequence comprising a polynucleotide sequence having atleast 90% sequence identity to the polynucleotide sequence of SEQ ID NO:85.
 11. The vector according to claim 10, wherein the vector comprises apolynucleotide sequence having at least 90% sequence identity to SEQ IDNO: 86.